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					  Journal of Histochemistry & Cytochemistry
                  http://jhc.sagepub.com/




Increased accuracy of absorption cytophotometric DNA values by control of stain intensity.
                  D C Allison, P F Ridolpho, E M Rasch, R W Rasch and T S Johnson
                                 J Histochem Cytochem 1981 29: 1219
                                      DOI: 10.1177/29.10.6170668

                             The online version of this article can be found at:
                               http://jhc.sagepub.com/content/29/10/1219


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                                  Official Journal of The Histochemical Society




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               0O22-1554/81/12121910$02.50
               The        Journal              of Histochemistry                              and       Cytochemistry                                                                                                                                  Vol. 29, No.                       10, pp. 1219-1228,                             1981
               Copyright               ©       198       1 by       The         Histochemical                      Society,           Inc.                                                                                                                                                               Printedin                 U.S.A.




                                                                                                                                                                           II

               Increased                                                        Accuracy                                                     of                  Absorption                                                                  Cytophotometric
               DNA                                       Values                                       by                   Control                                                of               Stain                               Intensity’
               DAVID                             C.           ALLISON,                                 PAUL                        F. RIDOLPHO,                                                    ELLEN                         M.            RASCH,                                    ROBERT                                W.               RASCH,

               and               TOD                     S. JOHNSON
               Department                      ofSurgery,                  Albuquerque                       Veterans               Administration                              Medical             Center            and         The              University                     of New            Mexico,                 Albuquerque                          New
               Mexico             87108                 (D.C.A.;                P.F.R.),              Departments                     ofBiophysics                         (E.M.R.)                and         Physiology                    (R.W.R..                          East       Tennessee                 State          Uniz’ersity.
               Johnson              City,            Tennessee,                 and         Life      Sciences             Division,                Los         Alamos              Scientific                Laboratory                    (T.Sj.).                  Los         Alamos,               Neu’          Mexico


               Received                  for         publication                   December                        23,        1980           and          in     revised                 form          April          6,      1981;             accepted                        April (OA
                                                                                                                                                                                                                                                                                        17, 80-334)
                                                                                                                                                                                                                                                                                             1981




               A method            of improving                 absorption          cytophotometnic                 cel- Cytometry          can also be performed                      at controlled           absorbance
               lular    DNA          values        by making              measurements              on Feulgen- levels             on autoradiographs                   of 3H-thymidine-labeled               cells to
               stained       cells     at optimal            stain     absorbances            has been          devel- allow       direct      study      of the DNA                content         of nonlabeled             Gil
               oped.     Stain      intensity         can be controlled                either      by alteration         GO and G2IM               cells.     Use of this technique                       on mixtures             of
               of the Feulgen                staining          reaction        or by selection                of “off-mouse          thymocytes,             spleen        cells,      bone        marrow          cells,     and
               peak”       wavelengths                of light           for   cytometry.             The       use of   liver   cells gave          essentially          identical          values       for G1IGO           cel-
               chicken         red blood            cells     as an internal             standard,          and of lular   a      DNA         content,         with       coefficients            of variation             of less
               computerized              cytometer             for the measurements,                     allows      se-than     3%.
               Iection      of the appropriate                   off-peak       light     wavelengths,             cor- KEY WORDS:         Absorption            cytophotometry;          DNA         content       (Feul-
               rection        for staining              variability          at different            sites     on the    gen);   Controlled           absorbance;           Stain      intensity.
               same    slide,      and rapid            calculation          of cellular         DNA         values.




                                                                                                                                                                                  off-peak                   wavelengths                             of        light              on         intensely                   stained                 slides             (14).
Introduction                                                                                                                                                                       Similar                  methods                    of      improving                               cytophotometry                    of    the             protein
Absorption                       cytophotometry                                       of      Feulgen-stained                                 cells             has         sev-stain              Napthol                   Yellow                       S        have               recently                 been            reported                         (29).
enal advantages                            for          the     determination        ofcellulan                                     DNA       content.          We found         that both                                                              measuring                             intensely                  stained                cells            at off-
Measurements                               can           be     made      on morphologically                                         selected       cells, peak      wavelengths       and                                                              measuring                             lightly            stained                 cells             near          the
such           as cells     in metaphase          or                                          infrequently                       occurring         tumor absorption            maximum                                                                 of           the          Schiff’s                reagent                   led          to         increased
cells.         The   slides    of Feulgen-stained                                                    cells               provide        a permanent        precision        in the values                                                                 of        nuclear                   absorbances                          obtained.                        Mea-
record       and                   can         be retrieved                                  for future      study.                                 In addition, surements                                          on       autoradiographs                                            of       H-thymidine-labeled                                 cells
measurements                                can   be made                                  on autonadiognaphs                                        of 3H-thy- excluded                                       S-phase                  cells             and              allowed                 accurate                    study                 of      the        cel-
midine-incubated                               cells,          allowing                    direct            study            of      the          DNA                 con-        lular          DNA               content                   ofGl/G0                             and          G2/M              cells         only.             In         addition,
tent       of        nonlabeled                          Gi/GO                   and          G2/M                 cells           (8).       However,                            in-
                                                                                                                                                                                   we        found                 that          the          use             of      chicken                    erythrocytes                            as          an         internal
strument                  glare,            diffraction                     at specimen                            interfaces,                     and           residual          standard      prior                      to slide preparation                                               ( 1 i ,28),    in conjunction                                       with
distributional                     error                can         lead           to        errors                in      nuclear                  absonbance                     a computerized                             cytophotometer                                                 (26),     provided       a rapid                                    means
values           (10,12,13,17,18,22,24,27).                                                 These              errors               are       especially                           for          selection                   of         off-peak                       wavelengths,                 gauging                    of         the          effec-
pronounced                         when      cells                   are intensely                            stained     (12).                      In order                      tiveness
                                                                                                                                                                                   to                         of      various                  understaining                                     procedures,                          correction                           for
increase                  the       accuracy                        of absorption                               cytophotometnic                     DNA                            staining                  variability                      at       different                         sites          on          a slide              (4),             and        calcu-
values,              we         investigated                  two          methods                     of      controlling                          the          inten-            lation              of      cellular                DNA                    values.                   The        cellular                   DNA                detenmina-
sity      of      Feulgen                   staining:                      1)     altering                   the         staining                  conditions                   tions
                                                                                                                                                                                  in     made                       with    this                     technique                       were  in agreement         with                                                flow-
the       Feulgen                  reaction                   to     produce                       lightly               stained             cells              for        mea- cytometnic                          measurements                             on                   the same    cell populations.
surement                   near             the           absorption                        maximum                         of       the        Schiffs                    stain
(560           nm);             and         2)          making                  cytophotometnic                         measurements                                  at
                                                                                                                                                                                   Materials                                  and                    Methods
                                                                                                                                                                                           Cell         preparation                          and          labeling.                       The       methodf preparing
                                                                                                                                                                                                                                                                                                         o                                           single-
       ‘Supported                      by the             Veterans                    Administration                          (D.C.A.)                    and         by        Na-cell         thymocyte                   and         spleen                 cell            suspensions                     from         outbred                   Swiss             mice
tional      Science                   Foundation                  Grant                 DEB        78-07753                      (E.M.R.).                                         has          been          described                     previously                    ).   (1
                                                                                                                                                                                                                                                                                For          labeling          in      vitro,    cells                were

                                                                                                                                                                                                                                                                                                                                                     1219



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1220                                                                                                                                                                                                    ALLISON,                          RIDOLPHO,                       RASCH,                        RASCH,                       JOHNSON




placed             in      complete                   Waymouth’s                          medium                    (1O’         cells/cc,               10%             fetal         calf
                                                                                                                                                                                         CA)          that      was       directly                 interfaced                     to      the           microdensitometer                  (Micro-
serum)              with           added              3H-thymidine                            (0.5          pCi/cc,               Amensham                         Searle,              products
                                                                                                                                                                                       Ar-                    and       Systems,                  Inc.,           Kingsport,                   TN)            (26).        For         flow-cytometnic
lington  Heights,     IL) under                  for
                                 5% CO2 at 37#{176}C 30 mm. Bone                                                                                                         marrow measurements                                 of      cellular                DNA               content,                 which             were            made          on       mith-
cells were    flushed    from femurs  with cold phosphate-buffered                                                                                                           saline namycin-stained                               cells         (9),         we         used           the         Los            Alamos               computer-based
(PBS).              Liver              cells          were             prepared                      by          pressing               of           tissue              fragments multipanameter                                 cell        sorter             (23)         with           excitation                  at      the       457-9              line,         at
through                 a 65-micron                         Teflon     mesh    into                              cold        PBS,   followed                             by sedi-140                mW        power.
mentation                 for removal                        of cellular    debris,                               and       by incubation                              in trypsin
(1%,         Grand                Island             Biological                   Company                    [GIBCO),                      Grand                  Island,             NY)
for 30 mm                       at 37#{176}C.
                                            Red                 blood             cells were lysed with NH4C1                                                      (0.17           M). Results
Cells were                       sedimented                      onto             slides  with a cytocentnifuge.                                                     On           some
slides,            a       trypan              blue            assay           for        cell            viability               (2)          was        performed                       Relative
                                                                                                                                                                                           by                   Absorption                                  by       Chicken                        Erythrocytes                                 and
immediate                        dipping               of      freshly               prepared                     slides           into          a       1:5           mixture             of
                                                                                                                                                                                          Mouse               Thymocytes                               at          Different                        Intensities                            of
trypan   blue (0.4%,        GIBCO)        and     PBS,    followed         by gentle                                                                              washing              of
the slides     in successive       rinses     of cold      1 % paraformaldahyde                                                                                             fixative
                                                                                                                                                                                          Feulgen                   Staining
(0. 1 M phosphate-buffered,           pH    7.2).    Chicken        erythrocytes                                                                               were            pre-       The         nuclear            absonbance                         for ten                     small             mouse      thymocytes                                       and
pared    freshly      for each        experiment          by
                                                         venipunctune            of                                                                       the            same             ten        adjacent             chicken                       enythrocytes                            was         measured         at                         560            nm
chicken.
                                                                                                                                                                                       (near  the absorption                                     maximum                          ofthe                 Schifrs      reagent)    on each
         Feulgen                  staining,                  autoradiographs,                                     and         cytophotometry.                                      The of 70 slides  stained                                  with   Schiffs                      reagent                  at different       pH values
slides        were               treated              with           a 3: 1 mixture                          of       ethanol                  and        acetic               acid    (pH
                                                                                                                                                                                       for   range,     0.6-1.4)                                        and             for          different                     time           intervals                    (2-60
 1 5 mm and placed                through        two successive              5-mm       washes       with each of        mm). Nuclear              absorbance             was      recorded            in units        taken                                                                                                                             directly
the following            solutions:          100%        ethanol,        95%        ethanol,       80%       ethanol, from the cytophotometer.                          On      faintly        stained         slides       in                                                                                                                      which
and distilled        water.         Hydrolysis          was performed               in 4 N HCI with poly- the                    enythrocyte             absorbance              was      less      than        4 units,                                                                                                                            the   rela-
ethylene        glycol      (PEG,         mol wt 6000,               Sigma      Chemical          Co., St. Louis, tionship               between             erythnocyte              and       small        thymocyte                                                                                                                                   absorb-
MO) (15 g PEG/SO                      cc 4 N HCI)               for     1 hr at 28#{176}C      (21).     The      slides ances      was       nonlinear            (Figure          1A).       On        lightly        stained                                                                                                                              slides
were     rinsed        in Schiff’s          reagent        (Sigma        Chemical          Co.) at the pH at
                                                                                                                         with      chicken           erythnocyte               absonbances                 between             4                                                                                                                         and             12
which      staining        was      to take       place.       Feulgen        staining       followed          immedi-
                                                                                                                         units,        however,               this       relationship                 was         linear,                                                                                                                               with              an
ately    for various            times       at room          temperature             in either        pH-adjusted
                                                                                                                         enythnocyte:thymocyte                   absorbance             ratio      ofO.37.          On the                                                                                                                            more
(4 N HCI) or unaltered                     (pH      1.4) Schiff’s         reagent.       The standard            con-
                                                                                                                     for intensely
dition     of Feulgen             staining       was      incubation          of hydrolyzed             slides              1
                                                                                                                                          stained        slides,      including          those       stained         under                                                                                                                          standard
hr in Schiffs                      reagent              at pH                   reagent, conditions,
                                                                           1.4. To remove                      the    erythrocyte
                                                                                                                      unreacted         nuclear        absonbance
                                                                                                                                                 Schiff’s                  was      greaten
we        placed            the        slides           through                three     than     20 units,
                                                                                                   5-mm            and the enythrocyte:thymocyte
                                                                                                                   changes                of     freshly    absorbance         ratio
                                                                                                                                                                         prepared
S02-water,      followed     by successive      rinses in distilled     water,    graded was      0.47      because        of the   relatively         low     nuclear      absorbance
alcohols,   and xylene.       For autoradiographs,       NTB-2      emulsion-coated      measured          for the intensely         stained        small     mouse      thymocytes.
slides (Eastman       Kodak,   Rochester,     NY) were exposed        for 2 to 4 days.        To     determine           whether    the      accuracy        of cytometnic            values
Cover              slips          were              mounted                 with              Cargille                Immersion                     Oil
                                                                                                                                                 (nD200            C
                                                                                                                                                                                          for       cells      of all         sizes             was          dependent                         upon               the         intensity                 of      stain-
1.524,             Vickens                  Inst.       Ltd.,           York,             England).
                                                                                                                                                                                          ing, we            used        mouse                   thymocytes                             labeled                   in     vitro            with          3H-thy-
         Cytophotometny                              was           performed                       with           a Vickers                    M8S                microden-
                                                                                                                                                                                          midine,              added                chicken                  erythnocytes,                and            prepared                      slides            that
sitometer                   (Vickers                   Instruments                           Malden,
                                                                                          ,Inc.                         MA)             with           a 400-700
                                                                                                                                                                                          were          Feulgen               stained                  as follows:                      Schiff’s                  reagent                 at pH              1 .4 for
nm photomultiplien                                    and 100x       oil immersion       lens (NA                                                              1.25).             Point
absorbances      were                                measured with     a 0.4   pm spot     utilizing                                                          a        20190              3, 4, and            60 mm;         and                       Schiffs                 reagent                  at     pH  1 for                        5, 7.5,   and
entrance    slit setting,                              giving  an estimated     spectral     bandwidth                                                        1 7 of
                                                                                                                                                                   nm                       10   mm.          Autoradiognaphs                              were                 prepared                      for one-half                          of the
for measurements                                     performed     at 560 nm (7,19,             personal                                                            slides
                                                                                                                                                                       communi-in each     staining         group.       The                                                                          nuclear      absonbance                                         was
cation,                Vickers                 Instruments                           Inc.).              Integrated                     nuclear     absorbances     measured        at 560    nm for a minimum                                                                                      of4O      Gi/GO       thymocytes
were          obtained                       from             50,000                 point               absorbance                     measurements            per-and     24   chicken       enythnocytes      present                                                                           in         3        random                   Polaroid
formed                  over,          or       corrected                   to,       a       I      x       I     scanning                  grid.            No          electronic photographs                       pen           slide.            The          picogram                       DNA                  values             for       the         thy-
corrections                      for  made except
                                            glare      when we performed
                                                            ( 1 were
                                                                7)                   ex- mocytes        were     calculated            with                                                                                                                                     the erythrocyte                                   transform.                      The
peniments      to determine       the effectiveness     of glare    corrections.       The
                                                                                            results   of these        analyses          are                                                                                                                                    shown     in Figure                                  1B, where                       the
Vickers     M85 microdensitometen          utilizes an electronic    masking      system
                                                                                            thymocyte      Gi/GO          coefficients                                                                                                                                            of         variation                   are         plotted                 against
for selection    of slide areas for cytophotometry.           No attempt       was made
                                                                                            the average       nuclear        absorbance                                                                                                                                                 values                measured                      for         chicken
to minimize      the mask size; an A-S or A-6 mask was generally                      used.
Nuclear                 absorbance                     was          measured                      without             subtraction                      of background,                     enythrocytes                 present                    on         the         same             slide.              Figure                 lB         shows             that

unless             a correction                        for         glare          was          being             used.           All       cells          included                 in the coefficients
                                                                                                                                                                                        a                       of variation          for the DNA       content       in Gl/G0
Polaroid                  photomicrognaph                           were           measured,                       without                any          morphologic                    thymocytes          of all nuclear        sizes    are at a minimum         in the linear
selection                  or      exclusion                   of      cells         from             measurement.                                                                    portion       of the curve         of enythnocyte         and  small    thymocyte        nu-
         Cellular                DNA                values           were            calculated                    with          an “erythrocyte                               trans- clean    absorbance         values     (Figure       1A).
form”:             target              cell          nuclear               absorbances                           were            divided                by         the         average
nuclear                 absorbance                     of       adjacent                  chicken                  enythrocytes                         and             then          mul-
tiplied            by       an         appropriate                      constant                    selected                on       the         basis             of       the          Selection
                                                                                                                                                                                      slide                          of Light                     Staining                             Reactions                         and              Off-Peak
processing                      and         measuring                   conditions.                       For         point          absonbance                          measure-
                                                                                                                                                                                          Wavelengths
ments,             the           scanning                   spot        was          placed                over           the       center               of        the         nucleus
being         studied,                   and          percent                  transmission                         was          recorded                     directly                   To
                                                                                                                                                                                      from       determine                    the          optimal                  staining                 conditions                        for        measurement
the       photometer.                          For      data           transformation                             and         storage,                we used        a SOL near the absorption                                                 maximum         of                            the         Feulgen                  stain            (560 nm),
system             III          microcomputer                       (Processor                     Technology                       Corp.,               Pleasanton,      we measured       the                                           nuclear     absorbances                                        of more                  than            40 G 1/GO




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CONTROLLED                                 ABSORBANCE                                      FOR                     CYTOPHOTOMETRY                                                                                                                                                                                                   1221




                                                                                                             C/)
                                                                                                              uJ
                                                                                                                                                                                   A.       PROPORTIONALITY                         OF       NUCLEAR                  ABSORBANCES                     OF            CHICKEN
                                                                                             0>-
                                                                                                                                                                                             ERYTHROCYTES                        AND          SMALL               THYMOCYTES                  AT     DIFFERENT               INTENSITIES
                                                                                                             0
                                                                                             06                                                                                             OF        FEULGEN                STAINING


                                                                                             <I


                                                                                                0-J
                                                                                                Cn<
                                                                                                m
Figure        1 Nuclear
               .                   absorbances             over         a                       <U,
                                                                                                                                                   .i.:                ,
wide      range        of Feulgen           stain     intensities
measured              at    560        nm.        (A)      Chicken                              LU
                                                                                                JC/)
erythrocyte/mouse                   thymocyte                mix-                               Ow
tunes      were        placed        onto 70 slides,           by-
                                                                                                Z2
drolyzed,             and      then       Feulgen            stained
                                                                                                LL0
under       different         conditions           to give van-
                                                                                                0
ations       in stain         intensity.           Nuclear            ab-                                    I
sorbance              measurements             were          pen-                               0F-

formed           on      10 small          thymocytes                and
adjacent          erythnocytes               for     each        slide.
(X) Trypan                blue         treatment.                   (#{149}) try-
                                                                        No                                                                                  ‘                               T                                    I                                    1                                  I                              T

pan      blue       treatment.            (B)       Slides         from                                                       )                             4                               8                                  12                                    16                               20                                24
chicken         enythrocyte/mouse               thymocyte
mixtures          were       Feulgen           stained          under
                                                                                                                    10
                                                                                                                                                                                    B.      RELATIONSHIP                     BETWEEN               COEFFICIENTS                 OF     VARIATION

                                                                                                                                                                                             (CV)      OF         G1/G0          THYMOCYTE                 DNA        CONTENT           AND
various       conditions,           and alternate               slides                                                                                                                      FEULGAN               STAINING             INTENSITY

were processed                 for      autoradiography.                                                                9
Nuclear         absorbance             for 40 G1IGO                 thy-
mocytes           of all sizes            and       24      erythro-
cytes     was measured                for each         slide.      Cel-                                                 8

lular     DNA         values      were       calculated            with
the     erythrocyte             transform.             The        aver-
age coefficients                of variation               forhe
                                                              t                                                          7

DNA          content         of G1IGO                thymocytes
are plotted              as a function                      of enythrocyte                          C/)
absorbance           values,        with     the number               of                            LU                  6
                                                                                                    I-
slides      studied         at each          stain      intensity                                   >.

shown        in the error            bars.      It is apparent                                    0
                                                                                                    0
that      a linear          relationship             exists          be-                                                5

tween        erythnocyte              and      small       thymo-                                   >-
                                                                                                    I
cyte     absorbance             on slides          with       eryth-                                I-
                                                                                                                        4
rocyte      absorbances             of8     to 10 units           and                                    0
that the coefficients                ofvaniation            for the
G1IGO          DNA           content          of all thymo-
                                                                                                                        3
cytes      are     at a minimum                   at this        stain
intensity.                                                                                        U-
                                                                                                  0
                                                                                                                        2
                                                                                                    >
                                                                                                 0




                                                                                                                                                            4                ,              a                 ‘                                       ‘               16                                       20                        24
                                                                                                                                                                      ERYTHROCYTE                                         NUCLEAR                                ABSORBANCE




thymocytes                      in     three               random              fields            for              six        slides            in each            staining           In         a separate                          experiment,                             slides            containing                      a mixture                 of
group             illustrated                   in         Figure              lB.        The                    DNA                  content               was             3H-thymidine-labeled
                                                                                                                                                                       calcu-                                                          thymocytes                                and      chicken     enythrocytes
lated         with         the         enythnocyte                        transform                              (see             Materials               and         Meth- were             hydrolyzed                          under     standard                             conditions        and stained                                  as fol-
ods).         The        coefficients                          ofvaniation                      ofthe                        Gi/GO               cellular              DNA lows:                Schiff’s                  reagent,                   pH             1.4,         for    mm;
                                                                                                                                                                                                                                                                                         60              Schiff’s             reagent,
content  averaged                                about               3%          for        thymocytes                                   stained                for        5 pH
                                                                                                                                                                              or           1.4,        for         mm;
                                                                                                                                                                                                                   4                and            Schiff’s                reagent,                 pH         1,      for      5 mm.              An
7.5 mm in Schifrs                                    reagent              at         pH         1 and                        measured                  at       560           autonadiognaph
                                                                                                                                                                             nm                                              was          prepared     from                              a slide    stained      for
                                                                                                                                                                                                                                                                                                                 mm.                           4
(Table            iA).           For      slides               processed                   for               autoradiography,                                   staining Nuclear                      absorbance                          was measured                                 at different       wavelengths                                   of
in      Schiffs            reagent                    at     pH           1 for            10
                                                                                          mm             also                gave         coefficients                       light          for       at     least             60        cells            in      three            contiguous                       microscopic                    fields
ofvaniation                     ofGi/G0                        cellular              DNA                     content                    that       averaged                   for
                                                                                                                                                                             less        each         staining                 group                 (Figure                 2).       We           found             that       the         absorp-
than         3%          (Table                iB).                                                                                                                           tion          maximum                           remained                           approximately                the            same            (560-580




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1222                                                                                                                                                                                              ALLISON,                        RIDOLPHO,                            RASCH,                      RASCH,                      JOHNSON




Table                 1. Nuclear                   absorbance                   values               after             various               types             ofFeulgen                   staining,               measured               at        560        nm       and           at      off-peak                    wavelengths                   of
lighr
                                                                                                                                                                                                       Schiff’s           at pH     1.4                                                Schiff’s           at   pH          1

                                                                                                                                                                                                                                          60 mm
                                                                                                                                                                                        4 mm                       60      mm                    (off      peak)                     5 mm                           7.5     mm mm
                                                                                                                                                                                                                                                                                                                             10

A. No autoradiographic              processing
  Average      chicken     erythrocyte         absorption       value                                                                                                                   6.3 1                     23.96                   8.82                       5.39                     10.     16                       12.29
  Coefficients       of variation       of G1IGO         thymocyte                                                                  DNA                 content                         2.84%                       8.33%                           2.83f                           3.OO’                           3.11’ 3.76%
B. Autoradiographic            processing
  Average      chicken     erythnocyte         absorption       value                                                                                                                   4.61                       20.78                  9.13                           -                         5.90                         7.92
      Coefficients                      of     variation              of      G1/GO                  thymocyte                        DNA                 content                       6.19%                     4.50%                   2.02%                          -                         3.48%                        2.95%

       ‘Nuclear                absorbances                  for     more         than           40     Gi/GO                 mouse            thymocytes                    and      adjacent          chicken      erythrocytes         were    measured         on six slides      in each     staining                                                 group   near
the      absorption              maximum                   of the          Feulgen          stain           (560         nm).         In addition,                  standard-stained                   slides   (60 mm
                                                                                                                                                                                                               in Schiff’s       at pH    1.4) were      measured       at an off-peak       wavelength
(-600-630nm)                     selected              to give       an     erythrocyte                 absorption                     value            of 9 units            (read    directly                                      T
                                                                                                                                                                                                          on the cytophotometer). hymocyte           G 1/GO       DNA      values    were    calculated
with     the          erythrocyte                  transform.              The      nuclear             absorbances                       and           coefficients             of variation             were    averaged         for each    of six slides       in each    group.




nm)          under                all        of       these               staining               conditions                            (Figure                     2A,B,D),          the        off-peak             wavelength                         had        point            absorbances                            between                0.1S
and        that          autonadiographic                                    processing                           did         not        greatly                    alter       theand  0.70,  which     are    well   within                                                 the       accurate                    measuring                     range
absorption                       curve               of      the          lightly           stained                     cells          (Figure                     2C).        Theof the cytophotometen.
coefficients                        of      variation      for                           the   lightly      stained     Gi/GO                                                  cells To test whether         the chicken                                               erythnocyte:G                           i/GO              mouse            thy-
measured                    near          the absorption                                 maximum         (Figure     2B,C,D)        were mocyte                                                          absonbance                       ratio            remains                  stable            over                off-peak               wave-
considerably                           lower       than  those                             for cells   stained      under     standard lengths                                                          of light, we Feulgen       stained                                                a slide              of      chicken                  eryth-
conditions                      (Figure                   2A).                                                                           rocytes                                                        and mouse   spleen   cells     under                                               standard                   conditions                    and
     Measurements                                     made           on the                standard                      stained       cells                     at longer measured          the     nuclear                                        absonbance                       of       more                  than             30       Gi/GO
off-peak      wavelengths                                         showed                  decreased                        variability                         in Gl/GO spleen        cells     and adjacent                                              enythrocytes                        at     560,             584,             605,       620,
nuclear                absorbance                          (Figure             2A).             We           also           found                 that         very    small and  625      nm wavelengths                                                   (Figure             3B).     Only                      cells           with   point
shifts           in       off-peak                    wavelengths                          within                  the          600-640                          nm range absonbances            within      the                                        effective              measuring                         range             of the     cy-
caused           large     changes                              in the          nuclear                  absorbance                         values           of stan- tophotometer             (0. 15     and    0.70)        were       measured;          in   order        to
dard          Feulgen-stained                                     cells.        Since                 the stain                       intensity             can vary  meet       this criterion          it was necessary              to scan a large         number          of
from           slide              to         slide          even             with           staining                        under               indentical        con-cells     at wavelengths              near     the     absorption           maximum.          Cellular
ditions                (22),         it is necessary                          to select                     the      precise                    off-peak       wave- DNA          content        of the G i/GO          cells was calculated            with the eryth-
length                that          will bring       the                     absorbance                            values                   of the cells         on rocyte
                                                                                                                                                                         a          transform;         the same          constant        (2.6)     was used        for each
particular                     slide          into          the       accurate                   range                 of       the          cytophotometer.          measurement               group.       The    average        of the G i/GO            DNA        values
In       practice,                     selecting                   this       off-peak                       wavelength                                for         nontrans-         measured                   at each   wavelengths                                  was
                                                                                                                                                                                                                                                                        7.04            ±      2.96%             (coefficient
formed           cells turns                            out to be                        quite    simple.                            The           wavelength                        of variation).
                                                                                                                                                                                      of                             It is apparent                             that        the            chicken                 erythnocyte:G1/
light         to be used                              is adjusted                         by trial     and                          error           in the                  longerGO            mouse             absorbance          ratio                        is quite            stable    over                       the 560-62                       5
off-peak                   ranges                    (600-640                       nm)              until              a      chicken                       erythrocyte          nm            range           of light     wavelengths                             when             cells   of point                        absorbances
absorption                    value    of                   9 units                 (taken             directly                       from               the cytopho- between                              0.15 and 0.70       are                             measured                      and          that       chicken                     enyth-
tometer)                   is obtained                       (Figure                   1B).           Application                             of          this method rocytes                            can be used   effectively                                  as an                 internal               standard                     for off-
to       standard-stained                             thymocytes                          gave              coefficients                           of         variation              peak          measurements.
of       Gi/GO                 DNA     content                              of less              than   3%    (Table                                         1).
         Figure                3A  shows       the                           effect               of the   off-peak                                           wavelength
                                                                                                                                                                                     Determination                                of Constants                               for       the           Erythrocyte
measurements                                  on          the       point                absorbance                           (stain                   intensity)                 and
nuclear                 absorbance                          (DNA               content)     of Gi/GO           thymocytes.
                                                                                                                                                                                     Transform
                                                                                                                                                                                      A
slide with  a mouse       thymocyte                                              and chicken          enythrocyte         mixture The        chicken        erythrocyte             transform       provides        a method      of con-
was Feulgen     stained       under                                           standard        conditions,         and     100 Gil    recting        for staining          variability         at different        sites   on the same
GO thymocytes         and    adjacent                                             chicken       erythnocytes          were      pho-slide      (4) and       allows        the calculation            of cellular       DNA      content
tomapped.                        The      nuclear                         and point       absorbances           for these            from
                                                                                                                                 cells          the     nuclear         absonbance.             We     calculated        the   constants
were      then                   measured         at                      560   nm (average            chicken      enythrocyte      needed         for this transform                 from     the nuclear        absonbances         used
nuclear                 absorbance                           of 22.5                     units)              and        at an                     off-peak                   wave-for Table                     ,1 according                   to       the        formula
length                of 630       nm                       (average                     chicken                   enythrocyte                          nuclear                  ab-
                                                                                                                                                                                  Constant                  =       (7)      x      (chicken                   erythnocyte:thymocyte                                        nuclear
sonbance                   of       9.5           units).           The             DNA               values                  were              calculated                     with                                                   absonbance                     ratio).
the        erythnocyte                             transform.                       It     is        clean              from             the             figure              that        a Table       2 lists   the   constants                                     for            the enythrocyte                                     transform
large         proportion                             of      the          thymocytes                          measured                            at         560       nm             that
                                                                                                                                                                                    had       generate         a mammalian                                      diploid               cellular   DNA                                   value7     of
point            absonbances                              greaten             than           0.9,            and             that           the          DNA                values pg/cell              (3,5,6)             for     slides                processed                   with           and             without                  autora-
of       these             cells             were            low.           Almost                    all         of        the         cells                measured                  at
                                                                                                                                                                                     diography,                   slides          stained                 at   different                   intensities                      and           measured




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CONTROLLED                                  ABSORBANCE                                    FOR                  CYTOPHOTOMETRY                                                                                                                                                                            1223




                                                                                                                     A




                                                                                                0
                                                                                                0




                                                                                                w
                                                                                              C.)
                                                                                                z
                                                                                                4

                                                                                              0
Figure            2. Nuclear          absonbances                              at dif-        Cl)

ferent            light   wavelengths             and                        intensi-           4
ties of           Feulgen      staining.       Slides                         of thy-
mocytelchicken                       enythrocyte               mixtures                       4
                                                                                              ‘U
were             Feulgen             stained             under               various          1

conditions.                  Three                   Polaroid
                                            contiguous
                                                                                              z
photographs            were    taken      for   each slide,
and      the       nuclear       absonbances             for     the
same       cells     measured         at different          wave-
lengths          of light.           Chicken
                               (#{149})              erythno-
cytes.      (0)    Thymocytes.             (A)     Standard
Feulgen              staining           (60        mm        with         Schiff’s
reagent            at pH             1.4).    (B)         Feulgen               stain-
                                                                                                                                                                                                             620  480
                                                                                                                                                                                                                           D
ing       with     Schiff’s           reagent            at pH               1.4 for
                                                                                                     z
4 mm. (C) Same              as B, followed        by au-
tonadiognaphy.             (D)        Staining      with
Schiff’s     reagent       at pH        1 for  S mm.       It
is apparent        that the coefficients       of van-
ation     of   nuclear        absorbances        of    the
                                                                                                         65
lightly            stained            thymocytes             are        lower
than       those          of    standard-stained                         cells                           56

measured              near      the      absorption                               maii-                  52

mum.        Off-peak           measurements                                   of the            ‘U
                                                                                              C.)         48
standard-stained              cells     in longer                            wave-            z
length         ranges        (600-620             nm)                         yielded         4          44

lower        coefficients           of variation.                                             4c


                                                                                              4          32

                                                                                                          28
                                                                                              ‘U
                                                                                              ._i        24
                                                                                              0



                                                                                                         12




                                                                                                                                              520          540         560        580          600                                            520      540           50            580           600

                                                                                                                                                    WAVELENGTH                                                                                 WAVELENGTH




at        560          nm,            and           slides              measured                         at       off-peak                      wavelengths            that      the      constants                were         generated                    by    measurement                           of all cells
(-600-640                     nm)           selected               to        give         chicken                    erythrocyte                           nuclear     in      a random                microscopic                   field,         regardless                of         the     point          absorb-
absorbances                      of         approximately                    9    units         per             cell.           The            constants               ance values.                   Thus,    to give               an average               7 of
                                                                                                                                                                                                                                                                 picognams                     per       mam-
for       cells       processed                    for     autoradiography                                     are       slightly               higher            than malian   cell,                  the  constants                  for very                   heavily           or         lightly          stained
those   for nonprocessed                                      cells              (Table        2). This                      is most     likely                      duecells contain                  a correction               factor for the optical      errors                                      of under-
to refractive     errors                                 between                   the       enythrocyte                         cytoplasm                            and ovenstaining.
                                                                                                                                                                        on                                    It can             be seen    that the    constants                                         varied    by
the       emulsion,                   although                some               modification                            ofthe            spectral                prop- 2-5%            even            for       slides         stained              and          measured                     under           optimal
enties            of the         Feulgen                 stain          by       autoradiographic                                     processing                can-conditions.                      Therefore,                 if more             accurate              absolute               DNA            values
not       be       ruled         out.         The         variation                 in constants                          for         cells          in different are      needed,                     the    use          of   an     internal               standard              in addition                   to      the
staining              groups              measured                      at       560       nm            is in pant                    due           to     the      fact               chicken               erythrocytes                      is necessary.




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1224                                                                                                                                                                     AWSON,                       RIDOLPHO,                              RASCH,                             RASCH,                        JOHNSON




               A
                                                                                                                                                         Table          2. Constants                     for the erythrocyte                                      transform  calibrated                                            to
                                                                                                                                                         give        a DNA      content                     7
                                                                                                                                                                                                           of pgfor  Gi/GO                                       mouse cell?

                                                                                                                                                                                                      No       autoradiographic                                                Autoradiographic
                                                                                                                                                                                                                   processing                                                         processing
                                                                                                                                                                    Chicken
                                                                                                                                                                  erythrocyte                        Number                                                            Number
                                                                                                                                                                   absorption                            of                       Constant                                     of                      Constant
I-
z                                                               .         .--:.:.:.:i..4-’s:-.                ;.                                                       value”                         slides                    ±   CV’ (%)                                     slides                 ± CV (%)
Lu
I-                               2.

                                                                                                                                                           5.5                                             6                    2.40±4
LI
                                                                                                                                                            7.0                                                                                                                 6                      2.70±9
4
z                        I
                                                                                                                                                          9.0          (off-peak’)                         S                       2.60      ±        2                         6                      2.75          ±      S
O
                                                                                                                                                          9.0                                              6                       2.43      ±       4                          3                      2.74          ±      9
<6                                                                                                                                                       12.5                                              7                    2.54         ±        S
-5
                                                                                                                                                         21.0                                                                                                                   6                          2.76      ±      3
-5
-5
Lu
                                                                                                                                                         24.5                                              6                        2.83     ±        8
LI
                                                                                                              ____________________                                 ‘Labeled          thymocytes              and      chicken            erythrocytes                          were         placed            onto          slides         and
                             ;                e           7           6                          5        4   -i;           ‘         .;                   Feulgen              stained      under       a variety              of conditions                     to produce                     different                stain        intens-
                                                                                                                                                         ities.       Alternate           slides      were          processed             for         autoradiography               and      nuclear              absorb-
                                                     POINT           ABSOR8ANtE
                                                                                                                                                         ances were measured            for at least 40 Gi/GO              thymocytes          and adjacent     erythro-
                                                                                                                                                         cytes per slide.      All except    the   off-peak        measurements             were   made     at 560     nm.
                                                                                                                                                         The constants        for the erythrocyte          transform        were      calculated    according      to the
                    B.
                                                                                                                                                         following   formula:      Constant  = (7)    X (chicken         erythrocyte:thymocyte          nuclear
                                                                                                                                                         absorbarice              ratio).  In the            erythrocyte              transform,                       the       nuclear           absorbance         for each
                                                                                                                                                         mammalian                cell is divided              by the       average          value               for         adjacent            chicken      erythrocytes
                                                                                                                                                         and         multiplied           by an appropriate                       constant                selected                  on     the         basis         of slide-pro-
                                                                                                                                                         cessing           and     measuring          conditions.
                                                                                                                                                               ‘In        units     read     directly       on the               cytophotometer.
                                                                                                                                                               ‘CV,           coefficient       of variation,               (SD/x)               x        100,          calculated                by       averaging                 of        the
                                                                                                                   T                                     constants               for all slides   in a staining               group.
                                                                                                                                                                   ‘Off-peakmeasurements         on standard-stained                                                   cells   (Schiff’s    at pH      1 .4 for 60
                                                                                                                                                         mm)     were     made      by selection      of the       wavelength                                               (-600--#{243}3Onm)        that    gave a
                                                                                                                                                         nuclear    absorbance     value   of approximately          9 units                                            for the chicken         erythrocytes.




                                                                                                                                                         Use of the Erythrocyte       Transform                                                                              for Measurement
                                                                                                                                                         of DNA    Content      of Gi/GO        and                                                                          Mitotic   Cells

I                                                                                                                                                        The
                                                                                                                                                         sion
                                                                                                                                                                       value   of the erythnocyte
                                                                                                                                                                        of nuclear    absonbance
                                                                                                                                                                                                                                        transform
                                                                                                                                                                                                                                      values      due
                                                                                                                                                                                                                                                                                 in eliminating
                                                                                                                                                                                                                                                                                 to staining
                                                                                                                                                                                                                                                                                                                           disper-
                                                                                                                                                                                                                                                                                                                      variability
                                                                                                                                                         on the same                       slide        (4) is shown                          in Figure                          4.        Alternate                        slides of
                                                                                                                                                         thymocyte/chicken                             erythrocyte                           mixture                           were           stained                      by the
w
LI                                                                                                                                                       standard        reaction      on lightly                                      stained.   Nuclear     absonbance       for
       __50                           sh          sb                 sb                              6o 6h      do              e.5                      cells    in four       random      fields                                    was measured        to provide     Gi/GO
                                                          E1.EdGTH                 Ir.N
               --                                    2S                                              ‘0         9s’5                                     DNA        values;       then   metaphase                                          and anaphase     cells   (M)   on all
                                            ERYTP0CYTE        NUcLEAR                 A8$ORBAPCE
                                                                                                                                                         slide            areas           were           measured.                         The                   nuclear                    absonbances                                   of         6
                                                                                                       chicken        erythrocytes              adjacent          to each        M cell were          also mea-
Figure      3. Relationship          between        nuclear     and point absorbances           at dif-suned,     which         allowed         use of the enythnocyte                    transform          to cal-
ferent      wavelengths        of light.       (A) Measurement            of nuclear      and point culate       cellular         DNA         content.          Coefficients          of variation         of 9.09
absorbances         of the same G1IGO                 thymocytes      at 560 nm (X, average
                                                                                                       for Gi/GO            cells      and      10.98        for M cells,           with     an M/Gi           DNA
erythrocyte        nuclear      absorbance          of 22.5 units)      and at 630 (#{149},
                                                                                         nmav-
erage enythocyte           nuclear      absorbance         of 9.5 units).    Cellular    DNA       val-content       ratio      of2.0i:i,            were       obtained         for thymocytes             stained
ues for the thymocytes             were calculated           with the erythnocyte       transform.     under      standard            conditions            and      measured           at 560      nm,       with   a
(B) Measurement             of the nuclear          absorbances of more     than     30 G 1IGO          10%    glare       correction      (17)     (Figure        4A).      The    enythrocyte         trans-
spleen     cells and adjacent          erythrocytes         with point absonbances         between
                                                                                                       form    did not lower                the coefficients               of variation         of the nuclear
0.15           and           0.70     at different            light              wavelengths                   (560,        584,       605,    620,      and
625           nm).           Cellular      DNA            content                 was calculated                    by          for
                                                                                                                            means        the
                                                                                                                                         ofthe    heavily        stained
                                                                                                                                                    eryth- absorbances         Gi/GO           and        M cells.           In
rocyte     transform       using a constant      of 2.6 for all measurements.              A large Figure         4B, the unconnected                        nuclear      absorbances               for the lightly
proportion        of thymocytes      measured        at 560 nm have point absorbances               stained         slide       measured             at 560         nm are shown;                the coefficients
greater      than 0.90 and these cells have low cellular                  DNA     values.      Mea- of variation                of the           Gi/GO          and      M cells         are      9.04         and       9.16,
sunement        at 630 nm shifts the point absorbance                  values of most of the
                                                                                                    respectively.                Measuring              the lightly         stained         cells      with         a glare
thymocytes          into the effective       range      of the cytophotometer             (0. 1 5 to
0.70)    and leads      to much    a smaller     variation     in measured      DNA        content. correction                1
                                                                                                                            ( 7) did         not     lower        these     coefficients              of variation.
When       cells with point       absorbances          between     the 0.15     to 0.70        rangeThe erythnocyte                     transform           of these       data for the lightly                     stained
were     measured         oven the 560 to 625 nm wavelength                      range      (Figure cells     (Figure           4C) provided                a distinct       improvement                   in the pre-
3B), the average          of the G IIGO cellular          DNA    content,    for all measured
                                                                                                    cision        of      the       nuclear          absorbance            values;         the       coefficientsof
groups,       was 7.04 pg ± 2.96%.
                                                                                                                                                         variation                  for     the       Gi/GO                 and          M           cells             were               lowered                    to         2.76             and




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CONTROLLED                                          ABSORBANCE                            FOR              CYTOPHOTOMETRY                                                                                                                                                                                          1225




                                A                                                                                                                      3.96,        respectively,                            with            a ratio               of      M        to         Gi/GO                      DNA               values
                  30
                                                                                                                                                      of        1.99:1.              The           results                were             similar               for          the          erythrocyte-tnans-
                                                                                                                                                      form      DNA                      values     obtained                             in off-peak                     measurements                                 on stan-
                                                                                                                                                      dard-stained                          thymocytes;                            the      coefficients                      of variation                             for the
                      15                                                                                                                              Gi/GO                  and M cells                           were  2.40     and                             2.86,   respectively,                                      with             a
                                                                                                                                                      ratio         of        M to Gi/GO                             DNA     content                               of 2.00:1.



 C’)
 -J
 -I
                       0               ttJL
                                                5
                                                    , , ,     -

                                                                    10               15
                                                                                           .

                                                                                                     20                                               Comparison                                of Flow-                      and               Absorption-
 uJ                                                                          DNA          CONTENT                                                     Cytophotometric                                        Cellular                      DNA                    Measurements
 0
 U-                                                                                                                                                   For        comparison                             of     cellular                  DNA                measurements                                    by        flow          cy-
 0                              B
                                                                                                                                                      tophotometny                               and           absorption                          cytophotometry,                         mixtures                     of
 w                                                                                                                                                    mouse                 thymocytes                         and           chicken                    enythrocytes                          were               treated                 in
                                                                                                                                                      one         of         two              ways:           1 ) The       cells                       were      placed                       onto           slides and
                                                                                                                                                      lightly            Feulgen-stained                        . Absorption                             cytophotometry                                     was pen-
 z
                                                                                                                                                      formed                  on all               cells           in      seven               random                    fields               at 560     nm,                        and
                                                                                                                                                      cellular                DNA                  values                 were              calculated                     with               the  enythrocyte
                                                                                                                                                      transform.                         2)     The           cellular   DNA                               content                    of 50,000        mithna-

                      0Tk4050                                                                                                                         mycin-stained
                                                                                                                                                      coefficient                        of
                                                                                                                                                                                                cells
                                                                                                                                                                                                variation
                                                                                                                                                                                                              was measured
                                                                                                                                                                                                                             for         the
                                                                                                                                                                                                                                                         by flow
                                                                                                                                                                                                                                                       DNA               content
                                                                                                                                                                                                                                                                                    analysis   (9,23).
                                                                                                                                                                                                                                                                                                    of
                                                                                                                                                                                                                                                                                                           The
                                                                                                                                                                                                                                                                                                             the          Gi/GO
                                                                   NUCLEAR                           ABSORBANCE
                                                                                                                                                      thymocyte                          peak            was              2.58%                  for         the          absorption-cytophoto-
                               C                                                                                                                      metric              values,                compared                          to      4      to       5%          for           the           flow-cytophoto-
                 60
                                                                                                                                                      metric                measured                         DNA                   content                  (Figure                    5).           The              ratios              of
                                                                                                                                                      chicken                 enythnocyte                          to      mouse                 thymocyte                          DNA               content                   were
                                                                                                                                                      0.35          and              0.36              for         the            absorption                       and              flow             measurements,
                 45                                                                                                                                   respectively.                            Thus,               the          two             techniques                          yielded                 comparable
                                                                                                                                                      results.



                 30
                                                                                                                                                      Measurement                                  of Cellular                             DNA                     Content                         Based                on
                                                                                                                                                      Two    internal                               References
                  15                                                                                                                                  To test the                        accuracy                  of absorption          cytophotometny             performed




                                                    JLA
                                                                                                                                                      at decreased                          Feulgen                  stain    intensities        with    application         of                                                     the
                                                                                                                                                      chicken                 erythnocyte                          transform,                      we        carried                  out           a series                 of     ex-
                      1)
                                                                                                                                                      peniments         in which    both                                      chicken        enythnocytes      and mouse      thy-
                                                                    1’O              15               20                                              mocytes       served     as internal                                        standards        for measurements        of the
                                                                     DNA            CONTENT                                                           DNA       content      of mouse                                         spleen,       bone     marrow,     and liver   cells.
                                                                                                                                                      Cells    of              each             type          were                either    mixed                        in     equal               numbers                     with
Figure           4.Measurement         of DNA       values       for G1IGO           and      mitotic        thy-
                                                                                                                                                      thymocytes                          on      left        alone                in suspension.                              Next,               chicken                   enyth-
mocytes          (M cells)     at 560      nm    on lightly         and   heavily         stained              rocytes
                                                                                                         slides.                                                              were              added                to      all         cell          suspensions,                           and            slides               were
Nuclear       absorbances       were    measured      for Gi/GO         cells     in 4 central         fields prepared                                                              and          Feulgen                    stained                under               standard                       conditions                         or
and     throughout         the   slide  for M cells.       Cellular      DNA          values       were       cal-
                                                                                                               lightly                                                   Feulgen                   stained.                 Nuclear                     absonbance                         was            measured                     for
culated               with             the      erythrocyte           transform.               (A)         GI/GO          and      M    nuclear
                                                                                                                                                      more             than              100      mouse                   cells         in random                      photographs                               from             each
absorbances                      measured         for    standard          Feulgen-stained  thymocytes        and
corrected                    for    10%   glare (17). The         coefficient          of variation     of nuclear                                    slide.           Off-peak                         measurements                       were            made                 on           the           standard-
absonbances                      for Gi/GO         cells    is 9.09,       that    for M cells      is 10.98,     and                                 stained
                                                                                                                                                     the                    slides,             and          cells         on        the         lightly           stained                   slides           were              mea-
M:G1/GO                    ratio             ofDNA                content          is 2.01:1.         The          erythrocyte         transform      sured            at          560         nm.           The           DNA                  values             were              generated                        with          the
applied      to cells      measured         in different     microscopic                                                   fields    did not       im-
                                                                                                                                                    erythnocyte                           transform.         All of the                                    coefficients                         of         variation                     of
prove     the    coefficients           of variation     of these        values.                                               (B)   Nuclear        ab-
sonbances       for     Gi/GO          and   M thymocytes,           obtained                                                  from     the  lightly
                                                                                                                                                    the Gi/GO                            DNA         content    for both                                    nonmixed                          and          mixed                cells,
stained   slide    without        glare    correction.     The    coefficient                                                  of variation      foras determined                                 by         the          off-peak                 method,                     were                less       than              2.7%
Gi/GO             cells            is 9.04,            that         for     M cells       is 9. 16, and                 the      M:G1IGO          ratio(Table               3).      Similar                 results               were            achieved                     when                lightly               stained
is     2.02:1.               (C)         Erythrocyte                   transform       of            the    DNA               values     in B. Thecells           were              measured                   at 560                nm,          with          all coefficients                            of variation
coefficient                   of        variation      of     the       DNA      content                for G1IGO                 cells  is 2.76,
                                                                                                                                                  being                less         than           3.8%.                 Since           the           addition                of      thymocytes                            to     the
that       for        M        cells          3.96,
                                              is            and       the M:G1IGO                     DNA      ratio            is 1.99:1.
                                                                                                                                                      bone             marrow,                    spleen,                 and           liver           cells          did           not           lead          to       any        ap-
                                                                                                                                                      pneciable                    spread              ofGi/GO                          DNA             values,t is apparent
                                                                                                                                                                                                                                                              i                                             that          the
                                                                                                                                                      Gi/GO                  DNA                 contents                   of          these            different                    cell          types             are         very
                                                                                                                                                      similar,                if    not          identical.




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1226                                                                                                                                                                                    ALLISON,                      RIDOLPHO,                                  RASCH,                    RASCH,                        JOHNSON




                  A                                                                                                                                                     Table        3. Off-peak     measurements         ofthe                                                   DNA                content                    of
                                                                                                                                                                        Feulgen-stained          mouse    thymocytes       and                                                  of bone             marrou’,                     spleen,
                                                                                                                                                                        and liver cells alone and             in mixture?

                                                                                                                                                                                                                                                                                                  DNA           content                  of
                                                                                                                                                                                                                              DNA              content              of                              Gl/GO       cells                   in
                                                                                                                                                                                                                                   GI/GO    target                                                thymocyte/target
                                                                                                                                                                                                                                   cells (pg/cell)                                                   cell mixtures
                                                                                                                                                                             Target          cells                                         ±        CVb                                            (pg/cell)                ±     CV


                                                                                                                                                                        Bonemanrow                                            7.05             ±        2.16%                                      7.04            ± 2.66%
                                                                                                                                                                        Spleen                                                6.99             ±        2.33%                                      6.76         ±        2.44%
Cl)                                                                                                                                                                     Liver                                                 6.77             ±        2.3391                                     7.12            ±            2.61%

‘U                                                                                                                                                                              ‘Mouse           bone        marrow,          liver,            and       spleen         cells      were          left     in suspension                            alone
0
‘4-                                                                                                                                                                     or     were      mixed    in equal        numbers           with      mouse       thymocytes;          chicken        erythrocytes
                                                                                                                                                                        were added,          and slides       were      prepared.            The    cells    were     hydrolyzed           and      Feulgen
                                                                                                                                                                        stained     under        standard         conditions.            Cytophotometnic      nuclear       absorbance            mea-
                                                                                                                                                                        surements      on more           than     100 mouse             cells    and adjacent         chicken        erythrocytes            per
                                                                                                                                                                        slide   were       made        at off-peak            wavelengths            ( -600-64Onm) selected            to give        an
         50
z                                                                                                                                                                       average      erythrocyte            absorption            value       of 9 units.        DNA        values       were      calculated
         40                                                                                                                                                             from    the erythrocyte               transform.
                                                                                                                                                                                ‘CV,      coefficient          of    variation,                (SD/x)       X    100.
         30


         20


         10
                                                                                                                                                                        content                 were           0.279              and               0.283            for         the         absorption                               and            flow-
                                                                                                                                                                        cytometnic                   measurements,                                   respectively.                      It is thus                     apparent                       that
                                                   10                     15                       20                   25                  30                          the           light-staining                   technique                      is compatible                                 with autoradiogra-
                                                       PICOGRAMS                               DNA                                                                      phy            and allows                   accurate                    measurement                             of        the Gi/GO        and                                  G2
                                                                                                                                                                        DNA              content                over          a wide                    range             of      DNA                  values.
Figure            5.         Comparison                  of        flow               and          absorption                    cytophotometry of
mouse             thymocyte                    DNA                 content.                   Thymocytes                         were            mixed            with
chicken             erythrocytes                       and       then    either      stained                             with           mithramycin                     Discussion
                                                                                                                                                                        or
lightly          Feulgen         stained.                    (A)    Flow    analysis      of                         thymocyte                and   chicken
                                                                                                                                                                     Several                   optical       problems,           including                                    instrument                          glare,      diffrac-
erythrocyte                   DNA            content.              The          chicken                 erythrocyte:thymocyte                                   ratio
is 0.35;           the         coefficient               of      variation                    of     DNA               content            for         the      G1IGO tion,              and         residual        distributional                                        error,      can                     cause      antifactual
mouse     cells is between         4 and 5%. (B) Absorption                                                                        cytophotometnic      dispersion        of DNA              values        obtained         by absorption            cytopho-
measurements           of the DNA      content   I of thymocytes,
                                                    78                                                                             with cellular        tometry         (1O,i2,i3,i6,17,22,24,27).                     These       errors         are     in-
DNA     content       calculated    by means   of the erythrocyte                                                                    transform.     The creased      when       intensely         stained        cells are studied.          There      is some
chicken      enythrocyte:thymocyte       ratio is 0.36,    and the                                                                 coefficient     of
                                                                                                                                                        opticalglare         in allabsonption               cytophotometens               (12,17,24).         The
variation               of     the       G1IGO           mouse                 cell         DNA              content             is 2.58%.
                                                                                                                                                        proportion         of glare         light      transmitted           to the recording             photo-
                                                                                                                                                                        multiplier                   increases                    with               increasing                    stain               intensity                        and           leads
                                                                                                                                                                        to      artifactually                  low         nuclear                      absorbance                         values                  for               intensely
Cytophotometry                               Performed                     on Autoradiographs                                                    of
                                                                                                                                                                        stained                cells.      Decreasing                                 the           intensity      of Feulgen                                                staining
Labeled                  Cells                                                                                                                                          therefore                  reduces        the                    error                due        to glare.   Indeed,                                          we         found
We            wished               to     determine                      whether                        we         could           obtain               accurate that electronic        corrections                                                   for     glare 7 ) ( did not
                                                                                                                                                                                                                                                                           1                               improve       the
values            for         the        DNA             content                      of      Gi/GO                 and          G2        cells          by per-precision      of cytophotometny                                                           of lightly       stained                        cells  (Figure                              4).
forming                  absorption                     cytophotometry                             on         autoradiographs                           of                      Similarly,                   diffraction                       at       specimen                    interfaces                         causes                    errors
3H-thymidine                            incubated       cells,                             thereby                 excluding                    the      labeled in absorption                              cytophotometnic                                      values     (12).    Diffraction                                          is more
S-phase      cells                   from      measurement.                                    Mouse                 thymocytes                       and Ehn-pronounced                                  at sharply       stained                              interfaces;       these     can                                 be       reduced
lich’s          ascites     tumor                       cells   were                        mixed          in            a 40-to-i                     ratio         and use
                                                                                                                                                                       by            of decreased                                 stain               intensity.
labeled            in vitro    with                     3H-thymidine;                                chicken              enythnocytes                          were        Finally,    distributional                                  error      due to heterogeneity                                                          of cellular
added,    followed                           by     slide            preparation                       and light      Feulgen                                   stain- constituents          (12,18)                               can be minimized              by means                                                       of multiple
ing. Autoradiographs                               were             prepared                        and cytophotometry                                         was measurements                  made                             with     “flying-spot”       cytophotometens,                                                  such
performed                      on        unlabeled                  cells             at      560            nm.        The            thymocyte                   Gi/as the Vickers              M85                             instrument            used in this study.                                                       However,
GO        DNA                distribution                    was      obtained                       from           measurement                              of cellssince               the         ideal           of     an           infinite                   number                   of          observations made
in       four          random                fields.            The             slide              was          then           scanned                  and          with
                                                                                                                                                                  mea-                 an infinitely                  small            spot             is not           attainable,                      all measurements
sunements                     were          made      on the                          large          Ehnlich’s    ascites                          cells.    Val-made                    with           flying-spot                  cytophotometens                             will        contain                    some                  re-
ues for            DNA                  were     calculated                            with          the erythnocyte                              transform.     sidual                   distributional                          error.                This            error           can            also            be         minimized
In addition,                            the   cellular     DNA    content    of                                                  the    mixture                      if
                                                                                                                                                                   was measurements                                 are made    at decreased      intensities                                                                    of      Feulgen
measured                      by         flow   cytometny.     For the samples                                                      measured                     withstaining   which                           increases    the optical     homogeneity.
absorption                     cytophotometny,                        we         obtained                     a ratio             of     G2           to G 1/                   The            extent      to which                           Feulgen                       stain            intensity                           can       be de-
GO DNA                       content     of              i.97            for      Ehrlich’s    ascites                             cells.   The                ratios creased                   for   absorption                           cytophotometny                                     is limited,                               however.
of thymocyte                       Gi/GO                 DNA                   content      to Ehnlich’s                               Gl/GO                   DNA At high                      transmission,                        the              light         absorbed                      by       chromophones                                       is




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CONTROLLED                                        ABSORBA                          NCE              FOR               CYTOPHOTOMETRY                                                                                                                                                                                            1227




so      small  compared                              to the                  background                              illumination                          that  it can-rocytes      as an internal                                        reference                   standard,                 select           the wavelength
not       be measured                             accurately.                    It has                been             shown     that                     the relative that    gives     an average                                           nuclear                  absorbance                     of         9 units    for                     the
photometric                             error             (RPE)               is given                    by         RPE LT/T
                                                                                                                      =                         In (lIT),                        erythrocytes,                        and       perform                    all subsequent                        measurements                              at this
where                   delta      T is           the          precision                     of        the            measurement                                 of     light wavelength                       (Figure               1). For               measurement                          oftissue                  culture            cells
transmission                       (T)          (20).           The           error               will          be            minimized                      at     In (T) containing                        many   lightly                       staining      cells,                   however,                 we found                     that
=       -     1, i.e.,            T       =       e , but                   will         increase                     at         absonbance                       values   selection                       of a measuring                            wavelength                          giving             point-absorbance
above               and below     0.434.                                    Previous                   work  has shown                                        that       quan- values    greaten        than                           1 5 for
                                                                                                                                                                                                                                       0.                   the        most          lightly             stained                cells       led
titative              nuclear absonbance                                        values                 can be obtained                                      from          Feul- to optimal       precision                                 of     cytophotometnic                                  values.
gen-stained                            cells        with              point               absorbance                              values             between                    0.1        The        off-peak                 measurement                              method                   has        the        advantage                      of
and    1.0 (27).    We                             have            found                  that          more     precise      DNA      mea- allowing                                                    an easy               correction                     for      cells        with          low        point-absorbance
sunements       can be                            made             when                 cells          with  point-absorbance     val-      values                                                  (<0.        15).           For         such             cells,           the         off-peak                 wavelength                      can
ues         between                     0. 15            and          0.70          are          studied                      (Figure               3). The              point- be changed                      so that             an absorbance                            value                    is
                                                                                                                                                                                                                                                                                            above 1 5 0. obtained.
absorbance                         values                for       most             cells           can          be           placed            into          this     range The  cellular                           DNA              value              can          then          be      calculated                     by      means              of
either              by light                    Feulgen              staining                     or      by          measurement                                 at appro- the erythrocyte                                   transform,                    with          a constant                   of      2.6         (Figure             3B).
pniate              wavelengths                        of         light.                                                                                                     Good    results                           are      also            achieved,                    however,                  with          the        light        Feul-
        The             excellent                  correspondence                                      between                      the        absorption                      cy-
                                                                                                                                                                                 gen-staining                       method.                 Light              staining              might             be      indicated                    in sit-
tophotometnic                                    DNA                  values                generated                             by          the          erythrocyte         uations    where       the use of chicken            enythrocytes         or of another
transform                        and      the                  flow          cytometnic                             values   obtained                                  for    internal
                                                                                                                                                                             the            standard        is inconvenient         or impractical,          or where        a
same      cell                    populations                              would        seem                         to validate      the                                        of
                                                                                                                                                                         use cytophotometen              is not available      for accurate         measurements          in
chicken                   enythrocytes                           as an             internal                reference                         standard                  for ab- the necessary         wavelength         ranges   ( 19). Simultaneous             cytopho-
sorption                       cytophotometry.                             The           erythrocyte                              transform                       allows         tometry                of      stains           specific                  for        non-DNA                      cellular                constituents
calculation                       of      cellular                DNA                   values            and       performed
                                                                                                                           corrects      on a Feulgen-stained
                                                                                                                                                    for      variability           cell might           also     preclude          the
in staining         intensity          at different           sites     ofthe        same     slide      (4).       use of appropriate
                                                                                                                   We                                   off-peak          wavelengths              for DNA               measure-
found       this correction                to be especially              important         when         diverse ments.            Under         these      conditions,            it would          seem        reasonable           to
slide     areas       must        be      surveyed          for     the      measurement of      infre-             stain     cells     to an optimal               density          (Figure , Table 1        1 ) and       to
quently         occurring            cell      subpopulations                 such      as mitotic            cells.carry      out    the       measurements                 near       the      absorption             maximum
The     variability          in the previously                reported           chicken       erythrocyte          rather      than      trying      to work          at precise           off-peak         wavelengths             in
DNA         values         (3,5,6,11,25,28)           may       be due           to differences               in order         to overcome              errors        caused        by heavy           staining.
measurement                techniques,             or there         may       be some        strain       differ-         The     use     of controlled              intensities          of Feulgen              staining        and
ences             in chicken                      cell         DNA                content.                Accordingly,                               the       constants         application                    of        the enythrocyte                              transform                   for absorption                            cyto-
for         the          enythrocyte                           transform                     need              to          be      standardized before                           photometry                          of     DNA      can                     be       applied               to      a wide     range                        of ex-
this       method       is employed.                                                                                                                                             penimental                    conditions.                     Stain              intensity                can be controlled                                effec-
        The    accuracy     of off-peak                                             measurements                                  on         Feulgen-stained                     tively     either                by light                   staining                 on by              the selection       of                         off-peak
cells         had              been            demonstrated                              earlier               ( 14),            and          our          results            con-light          wavelengths.                     The            technique                     is compatible                       with           measure-
firm          these              findings.                 We           found               that,          for           off-peak                   measurements                  ment            of 3H-thymidine-labeled                                      cells   processed                         for autoradiog-
at longer                       wavelengths                         (600    to                      620             nm),   the    coefficients                                    raphy
                                                                                                                                                                                   of              and thus     allows                          direct           measurement                            of G 1/GO and                          G2/
variation                      of nuclear                       absonbances                           for            Gi/GO     thymocytes                                   de- cellular
                                                                                                                                                                              M                DNA                                content.       The     use of two       internal                                                standards
creased                   to      less          than           3%,   compared                                  to      the 6%     value                            obtained gives     absorption                                 cytophotometnic        values       of mammalian                                                    cel-
when               the           same             cells         were    measured                                     at the   absorption                                 maxi-lular DNA         content                             with    a precision        identical   to that                                               for values
mum                (560-580                        nm,            Figure                 2A).            We  observed         less   improve- obtained         by flow      cytometry.           We                                                                                hope      that              the        future      corn-
ment     with                    off-peak                 measurements                                 of the same       cells     at shorter bination          of    other         corrective                                                                                      techniques                          (10,12,13,15,
wavelengths,                          with              coefficients                      of        5% at 520,        500,      and 480      nm17-19,22,27,29)          with     the     appropriate                                                                                     selection                     of stain        in-
(Figure                  2A).           This         may              be      due          to       the spectral      sensitivity        of thetensity     will lead    to further         refinement                                                                                       of      absorption                     cytopho-
microdensitometen                                  employed    (19)   on the                                          increased                     light            scat-       tometnic                values.
ten that     occurs                             at the shorten      wavelengths                                               (27).
        A single                  off-peak                     wavelength                         (490              nm)           had        been            employed            Acknowledgments
previously                         for           absorption                          cytophotometry                             (16).           However,                         The        authors            thank           Professor              Heuson             Suift.           Department                   of Biologj.                The
since             the          stain        intensity                  varies             considerably                              on        different                 slides, University                 of Chicago                for        his        valuable           advice         and        Mrs.          Elisabeth              Lanzl
even     under      standardized                                            conditions                         (22),              the         use of only                        f
                                                                                                                                                                               one or her         editorial           assistance.
off-peak       wavelength                                       for         measurement                                    of     all        slides is not                      op-
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