FLUOROMETRIC DETECTION OF DEOXYRIBONUCLEIC ACID SYNTHESIS; FOR

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FLUOROMETRIC DETECTION OF DEOXYRIBONUCLEIC ACID SYNTHESIS; FOR Powered By Docstoc
					          Journal of Histochemistry & Cytochemistry
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Fluorometric detection of deoxyribonucleic acid synthesis; possibilities for interfacing bromodeoxyuridine
                                  dye techniques with flow fluorometry.
                                           J Histochem Cytochem 1977 25: 913
                                                 DOI: 10.1177/25.7.70459

                                    The online version of this article can be found at:
                                       http://jhc.sagepub.com/content/25/7/913


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THE      JOURNAL              OF         HI5TOCHEMISTRY                 AND          CYTOCHEMISTRY                                                                               Vol. 25, No.               7, pp. 913-926,      1977
Copyright            © 1977             by The         Histochemical                    Society,   Inc.                                                                                                     Printed    in U.S.A.




  FLUOROMETRIC                                              DETECTION       OF DEOXYRIBONUCLEIC       ACID                                                                                               SYNTHESIS;
                        POSSIBILITIES                              FOR  INTERFACING       BROMODEOXYURIDINE
                                           DYE               TECHNIQUES        WITH  FLOW    FLUOROMETRY’

                                                                                                   SAMUEL                   A.       LA11
  The         Department                       ofPediatrics,             Childrens                  Hospital               Medical             Center            and         The          Center            for     Human              Genetics,
                             Harvard                   Medical                School,            300           Longwood                Avenue,            Boston,                 Massachusetts02115

                                                                         Received                  for      publication                January                 12,        1977


                  Fluorometric             detection           of the biosynthetic                incorporation            of 5-bromodeoxyuridine (BrdU)
             into deoxyribonucleic                     acid has permitted                cytologic        studies       of chromosome               structure,         replica-
             tion,     and repair.          Some        ofthese        phenomena,            previously         detected       using      BrdU-dye          techniques           on
             fixed       microscopic           preparations,              should      be particularly              amenable          to analogous             experimenta-
             tion     in fluorescence               flow      systems.         Problems          involved        in interfacing            BrdU-dye            methodology
             with      flow fluorometry                 are discussed.            The effects         of certain         chemical         modifications             of bisben-
             zimidazole            dyes      on their            spectroscopic            properties           and     potential          use    for      detecting           BrdU
             incorporation             into unfixed             cells are described.               Data    on the use and energy                  transfer         character-
             istics      of a pair of deoxyribonucleic                         acid-binding            dyes (33258          Hoechst        and ethidium              bromide)
             capable        of simultaneously                   providing        information            about      BrdU      substitution            and total        deoxyri-
             bonucleic          acid     content are presented.


       During     the                   past     few           years,    a number
                                                                             of meth- tum         yield       of this         fluorescence              is reduced              by
ods      for optical                      analysis                          acid
                                                                 of deoxyribonucleic   substitution             of     BndU            into      deoxyribonucleic
synthesis               have      been      developed.          These        methods acid.        33258       Hoechst         exhibits          at least      two      modes
utilize            the     incorporation             into    cells    of the        base binding
                                                                                       of                    to DNA,            neither         of which         appear          to
analogue,                 5-bromodeoxynudine              (BmdU),         which        involve          intercalation                (6, 53).        An    electrostatic
is then             detected         either       by changes          in dye           mode,
                                                                                    flue-            which          saturates             at   about       one      dye        per
mescence             (17,     28,     31,    46), by    Giemsa         staining        three       phosphate            groups,          is associated         with      little
(29,         30,      55)          or     by          immunochemical                      techniques                        fluorescence                       (40,        43).       At           higher               ionic      strength
(22).        Spectroscopic                            and        cytochemical            investiga-                         (e.g.         0.4M           NaCl,        pH                 7) A-T                    specific        binding
tions         have      contributed                              to the      understanding      of                         persists.                 Under      these                  conditions,                     binding         satu-
the         mechanisms                           by     which             these            techniques                      rates
                                                                                                                           de-            at         one    dye      pen                 3-4 A-T                    base       pairs       and
tect    BmdU         incorporation                                       (19,    43),   and     cytoge- produces                                     ahighly              fluorescent                    complex.                The         A-T
netic    applications             of this                               methodology          have       led
                                                                                                        specificity                                     of33258      Hoechst                       (14, 47, 67)                   probably
to    increased             understanding                                    of   chromosome            underlies                                     its application                           for differentiating                cen-
structure                   (38,          47),         replication            (32,       34),            and        me- tan              chromosome                               regions                in         the         absence              of
pair          (33,          54).          These                latter              studies                 have           also
                                                                                                                           BrdU           substitution                           (26).
suggested                a number                           of opportunities                       for            coordi-    Many                of the              spectroscopic                       properties                 of 33258
nating             techniques                          of     automated                      cytology                 withHoechst              are       shared              by      the         dye          DAPI          (4,6-diami-
those          for      optical                   detection                   of      deoxyribonucleic                     dino-2-phenylindole)                                   (45).            However,                      fluores-
acid   synthesis.                                                                                               cence       of    the        complex                                                of      DAPI      with                     poly
    A number                       of fluorescent                         dyes          have       been     used(dA . BndU)        is less        that                                    then           of the poly(dA. dT)
for detecting                        BrdU       incorporation                                into      deoxyn- complex         at pH 11, but                                              not       pH and
                                                                                                                                                                                                     7,        similar       pH
bonucleic                   acid.         33258       Hoechst                            constitutes           the
                                                                                                                dependence           characterizes                                                 the ability         of DAPI                     to
prototype                   of      agroup       of bisben.zimidazole                                dyes       detect     BndU       incorporation                                         into         cytological                 chre-
(49) which                   absorb      light                    near     350 nm                         and,       upon
                                                                                                                        mosome                  preparations          (46).                              By      comparison,
binding    to                deoxyribonucleic                        acid,    emit                       fluores-       33258             Hoechst        fluorescence                                  is sensitive         to BrdU
cence          peaking                    near          460        nm          (40,       43).            The       quan- at     pH 7 but               not          pH      (35,
                                                                                                                                                                             4          43). Acridine        orange,                           a
                                                                                                                         dye       which                 exhibits                    multiple       deoxyribonucleic
    I Supported                     by     a research                   Grant            (GM21121)                   and    a
                                                                                                                         acid       binding                 modes                   (5), has     also    been       used                           for
Research                Career               Development                  Award              (GM00122)
from         the      National                   Institute              of General                  Medical                 BndU
                                                                                                                          Sci-          detection.                        BmdU             substitution                      into    deoxy-
ences.                                                                                                                      nibonucleic            acid                   reduces              both               the      fluorescence
                                                                                                                     913




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914                                                                                                   LATT


intensity               (35) and            phosphorescence               lifetime                (19)     nical              obstacles                and           will           attempt            to       indicate
of bound                  acridine             orange.                 However,                   optimal some               potentially                  fruitful             areas           in      which            flow
results             with          cytological                   preparations                       require methods,                  together                with            BmdU-dye                 techniques,
extended                 illumination                      to “burn         in”               dull,          med
                                                                                                             might            be     used         to obtain                 basic        cytogenetic                infor-
fluorescence                  at sites              of      BmdU    substitution                           (17,
                                                                                                             mation.
18),       and          cytoplasmic                      fluorescence                   due         to     dye
                                                                                                                                     MATERIALS                    AND           METHODS
bound             to       ribonucleic                   acid        (57)        might             further
                                                                                                                       Dyes:       Ethidium               bromide           was purchased                    from Cal-
limit          the       use      of acridine                  orange             to detect               BrdU
                                                                                                                 biochem            and used without                       further          purification.               Con-
incorporation                      into       unfixed                cells.        Very         recently,
                                                                                                                 centrations                of the dye were                     calculated              from       absorp-
a small                  increase              in        cell         fluorescence                     due tion     to     measurements                        at 480 nm,                utilizing           an extinc-
BmdU              substitution                     has           been            observed                 when tion         coefficient               of 5685M’               cm        , based             on dry
cells         are stained              with           mithramycin                        or related              weight.           33258 Hoechst,                    as well as other                     bisbenzimi-
dyes          (58).                                                                                              dazole          dyes        (49), were               the generous                   gift of Dr. H.
      Alternative                 methods                   used          for        detection                 ofLoewe,            Hoechst            AG,         Frankfurt,                Germany.                An ex-
BndU             in cytological                     preparation                     employ              either tinction            coefficient             of4.2       10’ M
                                                                                                                                                                        x             cm’         at 338 nm in
Giemsa                 stain          (29,        30,         55)        (which             appears              pH 7 buffer
                                                                                                                   to                         (43) was used                   to determine                   concentra-
                                                                                                                 tions         of this           dye.        Roughly              comparable                  extinction
detect             selective               degradation                        of BrdU                  substi-
                                                                                                                 coefficients               were         determined                 for the other                   bisben-
tuted          deoxyribonucleic                     acid        (21)) or immunolog-
                                                                                                                 zimidazole               derivatives.                 7-Aminoactinomycin                   D was
ical       procedures                   (22).         The         latter          are       potentially
                                                                                                                 purchased               from        Calbiochem.                  Concentrations                     of this
adaptable                  to flow systems,                         in which              they        should dye were                   based         on an extinction                        coefficient             at 504
provide                a uniquely                     sensitive,                  positive               signal nm in pH 7 buffer                                           x
                                                                                                                                                            of 2.15 10 M’ cm1                          (20).
due         to BrdU.                Unfortunately,                   the       immunologi-                             Deoxyribonucleic                    acid     and polynucleotides:                            Calf
cal procedures                       all require                 deoxyribonucleic                acid            thymus,             C. lostridium               perfringens,           and Escherichia
denaturation,                    and        will          thus          not       be applicable                  coli deoxyribonucleic                             acid         were          purchased                 from
for studies                 requiring                live cells             or intact             chroma- Worthington                           Biochemicals.                   Poly        (dA dT) and poly
tin.                                                                                                             (dA BrdU)                 were        purchased              from P-L Biochemicals.
                                                                                                                 Extinction                 coefficients                used         for       deoxyribonucleic
      Some            of the        more           interesting                   possibilities                 for
                                                                                                                 acid       and         polynucleotide                    concentration                    determina-
 utilizing               BmdU-dye                techniques                     involve             fluomes-
                                                                                                                 tions        have        been        described             previously               (43).
 cence          activated              cell and              organelle               sorting             (2, 10,
                                                                                                                       Cytology:               Chinese             hamster               ovary           (CHO)           cells
 15, 23, 36). However,                                 effective             utilization                 of the  were        the gift of Dr. Arthur                           Pandee.           These         cells were
 ability           to detect             BrdU            in flow            systems             is contin- cultured                  in modified               (24) Hams             F-b         medium            supple-
 gent          on the            solution               of a number                        of technical mented                    with        10% calfsenum                    and 5% fetal                calf serum.
 problems                which          essentially                   constitute               the inter- BndU                 (Sigma)            and 8-methoxypsoralen                                (a gift of the
 face        between               BmdU-dye                  and         flow         methodology.               Paul        B. Elder              Company,               Bryan,             Ohio)         when         used,
 These             include             determination                  of the            quantita-                were       present            at concentrations                     of 5 x 10 and 1 x
                                                                                                                                                                                                    M
 tive         relationship                    between                    BmdU             substitution           10        M, respectively.                        Human             peripheral                lympho-
                                                                                                                 cytes,        cultured             in minimum                    essential            medium             sup-
 and            the          optical              signal (e .g . , fluorescence
                                                                                                                 plemented                 with        2
                                                                                                                                                       mM         L-glutamine                 and         20% fetal
 quenching),                    correction                 for the             effect        of cellular
                                                                                                                 bovine          serum,          were stimulated to divide                         by addition
 constituents                     other           that           deoxyribonucleic                acid
                                                                                                                 of phytohemagglutinin                             (32).        Specific            protocols             of
  (e.g. , ribonucleic                       acid,           protein)              on the             optical BrdU (1 x 10- M) or dT (1 x 10-sM) administration
 signals,               analysis              of BndU                   substitution                      under are        described               in the           appropriate                  figure         captions.
 conditions                  of changing                        deoxyribonucleic                 acid            Slides           containing                 metaphase                   chromosomes                     were
 content,              and,       when          unfixed                cells       are used,               oven-prepared               by air drying,                   while          CHO          monolayers                on
 coming              the permeability                           barrier            of cells           to dyescoven              slips         were          inverted              and         observed               as wet
 and         the        biological              effects              of dye            plus        light         mounts.
                                                                                                                  on                  Photographs                   were         obtained              with        a Leitz
 cells.                                                                                                          Orthoplan               microscope               using        either        transmitted                light
      Solution               of these               problems                  might            lead          to (Giemsa
                                                                                                                     a               staining)             or incident              illumination                  (bisben-
                                                                                                                 zimidazole               staining)             (37).
 number                of interesting                    applications                    of flow           tech-
                                                                                                                       Spectroscopy:                   Absorption                spectra           were        obtained
 niques            for studying                    mutagenesis,                        deoxyribonu-
                                                                                                                 with        a Cary            Model          118C recording                    spectrophotome-
 cleic         acid         replication                   kinetics,               X chromosome                   ten. Fluorescence                      spectra          were obtained                   with a Hita-
 inactivation                   and sister                 chromatid                  exchange                for-
                                                                                                                 chi MPF-3                spectrofluonometer,                        operated             in the ratio
 mation.              The       present             paper            will      describe             current mode.               Correction               of spectra            for wavelength                     depend-
 attempts                at overcoming                        some          of the above                   tech- ence       of instrument                     response             was essentially                    as de-




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                                                  BrdU-DYE                         TECHNIQUES                         AND            FLOW           FLUOROMETRY                                                              915

scribed        by Chen            (12). Fluorescence                        measurements            opposed            to the             blue        fluorescence                  of the          other
were       performed             either             in 0.5 ml,             5 mm          internal dyes),           exhibits            little        BmdU          sensitivity              at pH 7.
diameter          cylindrical           cuvettes            or in 4 ml, 1 cm square Addition                             of a lauryl                  chain         to the          other         end       of
cuvettes.           Values             of         the       fluorescence                of      33258
                                                                                                    the      molecule              (34630         Hoechst)             results          in a deriv-
Hoechst-deoxyribonucleic                           acid       complexes              in      the
                                                                                                    ative,         only         sparingly               soluble          in aqueous                  solu-
presence         of increasing               amounts            of ethidium             bromide
                                                                                                    tions,       that        is a very            poor        nuclear          stain,        presum-
were      corrected          both       for dilution              associated            with      ad-
dition       of the ethidium                     (a        13% correction)                 and by   ably       due       to a low               affinity          for deoxyribonucleic
screening          of exciting             on emitted              light      by the ethid- acid              (the      latter          inferred            from       the small              spectral
ium-deoxynibonucleic                   acid complex                  (a 6% comrec-                  change           caused            by the            addition           of a vast             excess
tion).      Quantum            yields          were        estimated           relativeo a
                                                                                       t            of deoxyribonucleic                      acid      to solutions              ofthe          dye).
value        of 0.51        for quinine                  sulfate         in 0.1 N H2S04,            Two         other            related             Hoechst             dyes,           33430          and
following          excitation            at 350 nm (13) Overlap                              inter- 33378,         possessing                 isopmopyl            on n-pnopyl               substit-
grals      between           corrected               energy        donor        fluorescence        uents,          respectively,                    exhibit           spectroscopic                   and
emission           spectra          and          energy          acceptor           absorption staining                      properties                    comparable           to        33342
spectra        were       calculated               by using           Simpson’s            rule     to
                                                                                                    Hoechst.              More            extensive               studies e.g.,        of the
approximate               the       integrals              in the           equation:J =
                                                                                                    effects         of long              term           exposure              to dye             on      cell
fF(A)e(X)X4dAIfF(A)dA,                   where          F(A) and #{128}(A) the   are
                                                                                                    viability           (25),        as well           as genetic              effects         such       as
donor      fluorescence             intensity             and accepten             extinction
coefficients           at wavelength                     (A). Ro, the critical                      sister
                                                                                                 dis-             chromatid                 exchange               induction              (54),       may
tance       for 50% efficient                      energy        transfer          was       calcu- assist        in the           choice         of the         bisbenzimidazole             de-
lated     as (8.78         x1025JQK2/n4)US                  cm (18, 61), using                  a mivative              most           advantageous               for      studies            with
nominal          value        of 1.5 for the index
                                               n,                          of refraction            unfixed           cells.
between          donor         and        acceptor             (59) and           an average             It is desirable                  that        bisbenzimidazole              staining
value      of 0.5 for K2 (see             text).                                                     ultimately               serve         as a reliable               quantitative mdi-
                                                                                                     cator       of BrdU                substitution                  in cells.            However,
                                            RESULTS
                                                                                                                             such        analyses              will           often           be      done         under             condi-
       While        33258           Hoechst                 has          been          the       bisbenzim-                 tions          of changing                        deoxyribonucleic                 acid           con-
idazole       dye             most      thoroughly                               characterized                              tent.
                                                                                                                           by               For    example,                         incorporation              of           BndU
spectroscopic                  techniques          and                           most      extensively                      throughout                 the         deoxyribonucleic                    acid        synthesis
used  to detect       BrdU                          incorporation            in                      fixed     cyto- (5)   phase        of                    the     cell     cycle    will                     result     in                in-
logic preparations,                               it may        not be the                           best     mem- creased         total                        deoxyribonucleic        acid                       content,
ben of this      chemical                            series       for studies                           with      un-which     can     be                    expected        to counter                        the reduction
fixed          cells.  The    terminal   substituent                                                   groups        in fluorescence
                                                                                                                     of                                            intensity                  due       to    BrdU-depend-
33258          Hoechst     are an N-methyl           pipemazyl                                                      group ent         quenching.                   If one            could          simultaneously             de-
and        a phenolic                     hydroxyl                      (49).         We        have         previ- termine       both                         BmdU    substitution                             and    total     de-
ously           described          (40)                a     number      of                     structurally        oxynbonucleic                               acid  content,                        this     feature       could
related           bisben.zimidazole                        dyes   in which                        the phe-          be converted                             from    a technical                          obstacle       to a po-
nolic          hydroxyl      is alkylated.                                      Insome       of these, tentially          useful     dual     parameter.            While                                                    the opti-
more             extensive       alkylation                                      of the     pipemazyl mal        solution        to this      problem           remains                                                      to be es-
group           is also present.         All                        ofthese        bisbenzimida-       tablished,            one    approach           involves          the                                                 simulta-
zole       dyes          exhibit             similar                  spectroscopic            proper- neous          use      of two      fluorescent             dyes,                                                    one      with
ties,          including                 sensitivity                      to       BrdU,                at          pH      BmdU-sensitive
                                                                                                                             7.                                fluorescence                           and           the         other
Subsequent                    studies               (1)          have           shown                that            33342 emitting                light        at      a         different             wavelength                     with
Hoechst             stains              unfixed                 cells       more           intensely                     and an      intensity             which             is independent                        of BrdU             sub-
evenly            than            does         33258               Hoechst                   (Fig.      1). Pme-
                                                                                                               stitution.
sumably,                   this           reflects                 a greater                         membrane     Past    experience                                          would                dictate            the        use           of
permeability                      for      33342             Hoechst.                                        33258   Hoechst       on a related         bisbenzimidazole       dye
   Addition                  of          small              apolar               groups         to    33258 as the    BrdU-sensitive          component           of such       a dye
Hoechst      improves                         the       staining                   of unfixed         cells, pain.  Possible       candidates          for the       BmdU-insensi-
but     more    extensive                            alkyl       on              aryl    substitution        tive  component          include        ethidium          bromide        and
does      not       appear               to be       advantageous.                               For exam- 7-aminoactinomycin                                                D,       both      of            which        emit
ple,     33770          Hoechst,        which      possesses                                     a biphenyl     light   at wavelengths                                             well    above              those      of 33258
terminal              group      (Table      I) and     yields                                  yellow    fluo- Hoechst     (20, 44,                                  65).         Of the      two,             ethidium        ex-
mescence              when                used             to       stain             intact                cells          (as
                                                                                                                            hibits          much           more              intense               fluorescence,                     which




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can     in          addition            be        excited            at      wavelengths                      also
                                                                                                                stant,        e.g.   ,   at    70   times      that   of the          dyes,  and         if
absorbed                 by        33258          Hoechst.                 7-aminoactinomy-                     the         amount             of     poly(dA . BrdU)     in         a mixture
cm D possesses                            a G-C binding                     specificity                  (5), with       poly(dA    . dT)     is progressively               increased,
which   contrasts                            with    the                   A-T       specificity              theof  emission           due      to 33258        Hoechst          decreases
33258           Hoechst               (14,     43,            67).          As       an     initial           markedly,
                                                                                                              ap-                while      that    due   to ethidium             increases
proach           to this              problem,                 the          dye        pain      of       33258
                                                                                                              very     slightly       (Fig.     2). The    ability       ofethidium                         to
Hoechst               and        ethidium               bromide                has       been          exam- serve      as an index              of total      deoxyribonucleic       acid
med.                                                                                                         content        seems        little     affected        by BmdU          substitu-
       The         fluorescence                   emission                spectrum                 of a mix- tion.      The      ratio         of the        two     peak       fluorescence
 tune         of      33258           Hoechst                and            ethidium                 bromide amplitudes            constitutes             a measure            of the        mole
 complexed                  with          polydeoxynucleotide                           contains              fraction        of poly(d.A . BmdU)          (Fig.         2, inset).        How-
 bands              peaking               near          460   and                 577   nm             (uncor-even,      at     least    with       synthetic              polynucleotides,
 rected),             reflecting                  the     two   dye                components.                this
                                                                                                                 If    curve        is concave         upward,             consistent                with
 the         polynucleotide                   concentration                     is     kept          con-     the       previous           observation         (43)            that          33258




      FIG.     1.     Staining               of    unfixed CHO    cells                  with  different                     Hoechst      bisbenzimidazole              dyes.    CHO    cells   were
cultured               monolayers
                      as                               on cover    slips                  in modified                    (24) Hams         F-b     medium          supplemented         with    10% calf
serum          and 5(7 fetal    calf                serum.    For each                   of the 6 dyes                    tested,    twenty-five         microliters         of a 1 mg/ml     aqueous
solution             of dye         was       added          to    a different                60    mm        petri       dish    containing           5 ml   medium.          One     and     one     half      hours
later,     the medium            was removed,             the cells were              rinsed       with      1 ml ofphosphate                buffered           M    (0.15
                                                                                                                                                           saline NaCl,
0.004    M KC1, 0.01 M Na                phosphate,          pH 7), and           the     covenslips          were     inverted         on slides,       mounted        in the  same
buffer.       lOLL fluorescent             polystyrene           spheres        (arrows)           (Particle       Technology             Corp.
                                                                                                                                      , Lot 795)       were added         as
internal        standards,          and cellular         fluorescence            was excited            by incident          light     (360-400       nm) and recorded         on Tn
x film      after    passage        through         a 460 nm barrier             filter.     Cells stained           with dyes 33342,              33430 and 33378 exhibited
bright       blue     fluorescence.            Those      stained        with       33258 fluoresced               blue      but less intensely,               those   stained    with
33770    fluoresced          yellow,        and those          stained        with       34630       exhibited        faint      fluorescence.          For all but these          last
cells,    the fluorescence               was localized            predominantly               to the nuclei.




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                                                              BrdU-DYE                    TECHNIQUES                            AND            FLOW             FLUOROMETRY                                                          917


                                                                                                             TABLE                      1

                                             SPECTROSCOPIC                                               AND                  STAINING                          PROPERTIES                    OF
                                                                SELECTED                              BIS-BENZIMIDAZOLE                                              DYES


                                                                                                               DNA               COMPLEXES                      cb

                                                                                                                                                            (dABrdU)n                 (C)            STAINING                   OF
                R                              ‘                                                     ABSORPT.                         FLUOR.                    (dAfl)                             UNFIXED                CELLS


      CH3-N(                         -    OH                              33258                              351                        460                          0.13                                        +

      CH3-N(                         -0C2H5                               33342                              352                        460                          0.11                                    ++

      CH3-N(                                                              33770                              355                        500                          0.58                                    +       +

  C12H5-N(                           -   OCH3                           34630           (d)                                             485                          0.71

  iC3H7-N(                           -OCH3                                33430                              348                        465                          0.13                                    ++

      CH3-N(                             -0C3H7                         33378                                352                        460                          0.11

          (I    Dyes    were               supplied                by Dr. H. Loewe,            Hoechst        AG, Frankfurt,         Germany.
          S     Measurements                       of           the      absorption     and      fluorescence         of soluble      dye-deoxyribonucleic acid   (DNA)                                                                and
polynucleotide                           complexes                  were       made  in 0.15 NaC1,
                                                                                            M               0.005 M Hepes,        pH 7.
   C Relative       quantum                                      of complexes       with      poly(dABndU)
                                                                                                     .           and poly      (dA dT) were          obtained     from                                                               corrected
emission      spectra     (12).                                 Classification      of the staining           of unfixed     cells was based         on photographs                                                                   such as those
ofFigure       1.
   I’ Binding       of 34630 to DNA                                               was         probably              incomplete.

Hoechst                       binds            more              tightly            to        poly       .   (dA
                                                                                                              BmdU)              this         estimate,               addition                of     aliquots              of     ethidium
than                   to       poly(dA            .   dT).           Analogous                      calibration                 to     complexes                    of     33258           Hoechst              with           an         excess
curves    with     material                                          of known              composition                        willbut     constant       amount                             of deoxyribonucleic                   acid
be needed      before       this                                     approach              could     be used                   forresults       in a quenching                              of 33258    Hoechst                     fluores-
analysis                      for        BrdU             substitution                        in     biologic               sam- cence    (Fig.    4). This     is presumably                                        due,           at least
ples.                                                                                                                            in part,     to energy     transfer,       and                                  is 50%           complete
    An                 additional                      feature       to            be     considered                       when at   ethidium/phosphate                                 ratios           near             0.03.            Up-
33258                  Hoechst               and            ethidium                    are    both                bound        ward
                                                                                                                                 to       curvature                         in      plots      of       Fo/F             versus             ethid-
deoxyribonucleic               acid     is the                                           energy           transfer              ium           saturation           may                 reflect           additional                    effects,
possible         between         the two dyes.                                             The      fluorescence                e.g.    ,     to     deoxyribonucleic                       acid          unwinding                       by
emission            spectrum          of 33258                                           Hoechst             overlaps           ethidium           (48)                 or     to      displacement      of  33258
the      absorption            of ethidium                                              (Fig.       3), fulfilling              Hoechst        from                   deoxyribonucleic          acid     by ethid-
one      of the       criteria      for efficient                                             energy          transfer          ium.     Preliminary                         measurements          based    on ab-
between                        the         two                (18,      61).        Under         the                 solvent sorption         difference                       spectra                indicate              that            bind-
conditions                          employed,                  the     critical        separation                        be- ing       competition                           between                  33258              Hoechst                and
tween                   the      dyes          for        50%          efficient               energy              transfer     ethidium                  for    calf        thymus                deoxyribonucleic               acid
(Re)             is approximately                                    25-30         A.2        Consistent                 with is very             small,        even              at high       total      dye:    deoxyribo-
                                                                                                                              nucleic              acid     ratios.               This    may       reflect      the fact                       the
      2        These          calculations                     utilized           a value            ofl.08           x 10 ethidium                     binds       to           deoxyribonucleic           acid    by in-
cm3      M       for the overlap         integral,           1.5 for the                                           index
                                                                                                                      (66), oftencalation
                                                                                                                                while          33258        Hoechst             binds
refraction,            0.5 ± 0.2 for the            orientation           factor,        K2,
                                                                                             along       the      outside       of the         deoxyribonucleic         acid
and 0.5 ± 0.1 for the quantum                          yield     ofbound          dye. A
value          of 0.5 is a good estimate                of K2 expected             for twomolecule               (6, 53).       Other          combinations                 of dyes
randomly             oriented     intercalating              dyes,     but oven esti- capable                of binding          to deoxyribonucleic            acid      and
mates            K2 expected         for one          intercalating              and      one potential
                                                                                             of                        use    in flow          fluorometry              are      pres-
nonintercalating              dye. Assuming              the latter        dye to lie ently             being        examined.
in a plane             tangent to the        deoxyribonucleic            acid       K2
                                                                                                 Once        appropriate            staining           conditions             for si-
will then            be approximated               by sin2 0, where
                                                   /2                           0 =
the angle            between       the second            dye’s emission              dipole multaneous               measurement                 of BrdU         substitution
and the deoxyribonucleic                       acid helix          axis.                     and     total      deoxyribonucleic             acid     content         can       be




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918                                                                                                             LATT


                                                                      lO                                                                                                                                                  m
                                                                                                                                                                                                                          w
                  MOLE             FRACTION                                 075
                  POLY            idA     BrdU)                                                                                                                                                                           >
                                                                                                                                                                                                                          w
      60

                                                                                                                                                                                                                          -4
                                                                              0           025    0.5 075     10
      40                                                                                                                                                                                                                  -4
                                                                                   MOLE     Fz N (dA BrdUI,,
                              I
      20
                          /        1’G9Q                                                                                                                                                                                  0

                      I
                  I                            .
                                     .
                                   #{149}.    #{149}i         .
                                                            #{149}
                                                                                                                                                                                                                          C)


        -   400                         450               500                     550                600
                                                                                                                                                           WAVELENGTH                nm
                                               WAVELENGTh                         (nm)
                                                                                                            3. Spectral          overlap
                                                                                                                              FIG.                 between           33258      Hoechst
    FIG.     2. Estimation                of BrdU          content         in polynucle-
                                                                                                fluorescence             and ethidium           bromide            absorption.        Flu-
otide       mixtures          using         33258         Hoechst          and     ethidium
bromide.           The fluorescence                 of mixtures              of 1.3 x 10 orescence                of a solution          1 x l0M in 33258 Hoechst
                                                                                                and 5 x 10- M in calfthymus                           deoxyribonucleic          acid
M 33258          Hoechst,           2.6 x 10 M ethidium                       bromide,
                                                                                                was excited             at 350 nm. The emission                         spectrum        was
and      2.0 x 10 ‘ M polynucleotide                          was excited            at 350
                                                                                  contained corrected
                                                                                                                   for wavelength                 variation            of instrument
nm.         The         polynucleotide                    component
                                                                                                response          (12).     The    absorption               spectrum           was      mea-
poly(dA       . BndU)         and         poly       (dAdT)
                                                        .          in the           ratios
shown.          As      the       proportion              of poly(dABrdU)
                                                                       .                in-     suned      on a solution          5 x 10M in ethidium                       bromide
                                                                                                and      1 x 10-s M in calf                 thymus            deoxyribonucleic
creases,         fluorescence              peaking           near      460 nm, to        due
33258       Hoechst,          decreases,             reaching            a limit         of lessacid.     The absolute           extinction            coefficient          is based      on
than       0. 15 that           of the         control.         At the          same            a value
                                                                                            time,             of 5685 M’ cm’            at 480 nm for the free dye,
                                                                                                determined            on a  dry weight            basis.       The overlap           be-
fluorescence             at 577 nm,              due       to ethidium              bromide,
does     not decrease,               but      instead          increases          by a few      tween       the two spectra              is shown             as a cross-hatched
                                                                                                area.     All samples           were dissolved               0.15 M NaC1,            0.005
percent.         The insert            depicts       the ratio           of these        ampli-
tudes       as a function                 of BndU             content.          In order
                                                                                       to
                                                                                                M Hepes,         pH 7at room          temperature            (approximately
reduce         the     relative          contribution              of 33258          Hoechst 22#{176}C).
fluorescence          to that       of ethidium             at the        dye     ratios
used,      vertically          polarized           exciting         light       (wave-                                        5                                                                 DNA
lengths       populating          the first         excited     electronic          state
of 33258 Hoechst              but a higher            excited      state      of ethid-                                       4                                                           ,,“_           dA   dTI,


ium),      was used,       while       fluorescence            was      measured
through          a horizontal            polarizer.          Spectra         were        not
                                                                                          U.3

corrected         for wavelength                variation          of instrument          ..
                                                                                          U.
response.                                                                                                                     2



established,                             it     should               then          be     possible         to     adapt
flow  techniques                                  both    to measure                           sister       chmoma-           0           .#{243}l   .62         .03             4        .05         .#{243}s m
tid exchanges                                  and     to isolate    late                         replicating             X                            ETHIIUM/PHOSPHATE

chromosomes.                                       While      neither         the                     biochemical             FIG.   4.       Quenching            of      the        fluorescence                   of        33258
mechanism                               non         the precise       biologic                       significance
                                                                                              deoxyribonucleic                   acid      complexes               by ethidium             bro-
of sister            chromatid              exchange              (S.C.E.)            forma- mide.        Aliquots            of 10 M ethidium                       bromide         were
tion     has       yet been determined,                   the correlation                  of added      to a solution                                   M
                                                                                                                                    of 2.0 x 10 deoxyribonucleic
                                                                                              acid and 5 x 10-vM 33258 Hoechst                                   in 0.15M NaCl,
S.C.E.          induction,              mutagenesis                and/or         carcine-
                                                                                              0.005      Hepes,         pH 7. The                  ratio       of the        fluorescence
genesis           by chemical               agents         (54),     as well           as the amplitude          without           ethidium            (F0), that
                                                                                                                                                          to            in the pres-
abnormalities                related         to S.C.E.’s              in “chrome-             ence      of ethidium,                  (F),     corrected             for dilution           and
some        fragility           diseases”          (3, 11, 41) is well                   docu-estimated          inner-filter              effects,        is plotted          versus        the
mented.                                                                                       ratio     of ethidium           to phosphate                for calf thymus,                 E.
                                                                                              coli,   and      C. perfringens               deoxyribonucleic                   acid,     as
     At present,                  S.C.E.’s           are       typically             scored well       as poly          (dA dT) and                  poly        . BndU).
                                                                                                                                                                (dA              In all
manually,               on photographic                   prints.         The       availa- cases       33258 Hoechst                 fluorescence              was excited           at 385
bility       of BndU-Giemsa                     techniques             for revealing          nm. Addition             of ethidium                resulted          in a reduction             in
S.C.E.’s         (e.g.,      Fig.      5) has       permitted             the     iitia-      33258       Hoechst             fluorescence.                 A 50%           fluorescence
                                                                                              quenching           of 33258           Hoechst            (F0/F= 2) occurred             at
tion       of procedures                   for      automatically         scoring
                                                                                              ethidium/deoxyribonucleic                         acid        ratios        near       0.03,
S.C.E.’s            directly          from        microscope               slides         (69).
                                                                                              varying        slightly           with       the type            of deoxyribonucleic
Chromosomes                     from        cells       which         have          divided acid used.




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                                               BrdU-DYE                       TECHNIQUES                      AND     FLOW               FLUOROMETRY                                                              919

twice           in      the  presence    of BrdU        will                                probably            sensitive
                                                                                                               be                       fluorescence).                     An         analogous               approach
difficult              to use for S.C.E.      detection                                     in flow         sys-utilizing                acridine                orange,                  rather           than          33258
tems,            since        S.C.E.’s               merely               interchange                     chre- Hoechst,                 has   recently               been                independently                   pme-
matids                in   these        chromosomes                         without                 altering     posed        by        Bender      (4).           However,                   the suitability                of
total            chromosome                      fluorescence.                However,             if
                                                                                                 acridine       orange        for such       a purpose          may      be lim-
cells           are     allowed                to undergo               three         cycles       of
                                                                                                 ited,    since    the     major     effects     on acridine            orange
BrdU             incorporation,                     S.C.E.’s         occurring           during fluorescence            due      to BmdU      substitution            requires
the       first   two             cycles     will         manifest        as nonrecipro-        prolonged          illumination.
cal      segments                  of bright            fluorescence            (Fig.      6, II). Analysis           of S.C.E.’s        by flow         fluorometny,          as
In      the      absence             ofS.C.E.’s                approximately                           half      of
                                                                                                                 described               above,           would            ideally             be     performed             on
the       chromosomes                       from        such          cells          will       show           twochromosomes                          from            a        population                  containing
dull          chromatids                    (Fig.       6,     III)        while              the       others only          third            division             metaphases.                         However,                 as
will          contain          one dull               and     one          bright              chromatid       long          as      one         is    dealing              only           with        a    mixture              of
(Fig.           6, I).       S.C.E.’s               occurring               during               the     third second             and         third       division               cells,        the     relative           con-
cycle          will        cause        only          a reciprocal                     intensity                 tnibution
                                                                                                                in-                      of     each          should             be       determinable                   from
terchange                  (Fig.        6, ID and      IE),                    but          such       events the      ratio            of chromosomes                              found              only    in third
should               comprise           only approximately                                   one-fifth        of
                                                                                                              division               cells (e.g. , Fig.                    6,     II and             III)   to those
the     observable         S.C.E.’s        in these       cells      (62).     Thepresent          in both        second       and      third       division      cells
remaining          events (e.g. , Fig.         6, II) will        contrib-        (e.g.,      Fig.     6, I). While           precise         quantitation             of
ute to a class          of chromosomes              with      fluorescence        S.C.E. frequencies                 from     flow      measurements          on
intermediate           between         the two extremes                 (Fig.      6,
                                                                                  such      chromosomes               might       require         involved        cal-
I and        III).  This      intermediate             class       should         be
                                                                                  culations,          they       would      present          two      advantages.
resolvable          with       a two         dimensional               analysis First,          a chromosome-by-chromosome                          analysis
(BndU-sensitive           fluorescence            versus         BmdU-in-         of clastogen            induction         of S.C.E.’s          would         be pro-



                              .         F



                                                                                                                      .J
                                                                           (

 H’s’                                                                 )                                                  t
 ,                                          ft
                                                                                                                                                                            0




 A                                                                         I                                     B
    FIG.   5. Induction        of sister       chromatid           exchanges         in CHO          cells      by 8-methoxypsonalen                  plus     light.       CHO
cells,   cultured      in the medium             described        in the caption            to Figure         1 were     exposed       to approximately ergs/
                                                                                                                                                     10’
mm2 of light,         predominantly             at 365 nm.           One hour          before    illumination,            the cell in (B) received 8- 10   M
methoxypsonalen,            while      the control           (A) received         none. hr after
                                                                                    1’/2                   illumination,                     M
                                                                                                                                   5 x 10 BrdU               was
added to the cultures,            and    the   cells    were      grown      in the      dark              C
                                                                                                at 37#{176}for 41 hr.         Slides     were      made      by air      drying,
stained      with 33258 Hoechst,             mounted        in 10 offresh
                                                                  M              33258 Hoechst                      M
                                                                                                           in 0.005 NaC1,          0.001 M KC1, 0.003 M
Na Phosphate,        pH 7, exposed           6 hrto light      from     a 20 Watt         cool white         lamp     at a distance         ofabout        6 cm,      placed
15 mm in 0.3 M NaC1.              0.03 M NaCitnate                               and
                                                               pH 7, 65#{176}C, stained                with       Giemsa       (37).




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920                                                                                      LATT


vided.          Second,            it   should         be      possible          to      isolate lymphocytes            are           allowed              to    initiate            a cycle              of
and       examine              biochemically                chromosomes                  in the
                                                                                              deoxyribonucleic                     acid          replication                in    the      pres-
intermediate,                   S.C.E.-containing               class.                           ence     of   BmdU          but          then        complete               replication                  in
    Isolation             of    late     replicating               X chromosomes                 the presence          ofdT     (The     “T-pulse”                          protocol),             late
would        exploit            observations                already       made           on      replicating
                                                                                                cy-                    chromosome          regions,                           especially
tological    chromosomes            preparations                             (42,       68)      those
                                                                                                to-          of the     X, will      contain        dT                      but      not      BmdU
gethen    with     the   previously           demonstrated                                  abil-and     fluorescence                brightly              against               a BrdU-sup-
ity of flow techniques           to sort      individual                              Chinese pressed           background                 (68).
hamster             M3-1    (23)           and        muntjac         (10)            chmomo-        While        normal              late       X         chromosomes                     should
somes        and     to resolve             at     least      23 different           peaks ultimately            be sortable,                           large      structurally                   ab-
in mixtures             of human                 metaphase              chromosomes        normal         X chromosomes                               should      present          an          even
(9).  For           example,     if              human            female        (46,       more
                                                                                         XX)          accessible      target                        for isolation.            Such            chre-




     FIG.    6. Sister        chromatid            exchanges        in 3rd division         cells.   Shown         are chromosomes               5 from human        lympho-
cytes       which       had      replicated          3 times      in the presence            ofM 10BrdU.        In the absence            of S.C.E.’s      occurring
during        the first two cycles               ofBrdU       incorporation,         halfofthe         chromosomes             in these      cells would     be expected      to
show       the bright-dull              fluorescence         seen in row I, while              the rest would            be expected          to be uniformly        dull (row
III).     S.C.E.’s        occurring          during      the first       two cycles       would      cause       nonneciprocal          fluorescence       patterns      in 3rd
division         cells (row     II) which          would     manifest        as intermediate            overall       chromosome           fluorescence.




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                                         BrdU-DYE                     TECHNIQUES                       AND       FLOW          FLUOROME’rRY                                                 921




    FIG.     7.                                 i; to   highlight       the   relative    fluorescence            of an isochnomosome for the     long
arm      of the       X. Lymphocytes         from        an individual          with   46, X, i(Xq)             cells   were   cultured       46 hr in medium
containing          BndU    followed   by a 7 hr pulse              of dT and the slides             stained       with   33258 Hoechst.        The isochnomo-
some      (long     arrow)    stands   out from           the other       chromosomes,           including          the early    replicating,       structurally
normal          X (short   arrow),   because          of both     its size and intense            fluorescence.



mosomes,            such     as isochromosomes                for the     long      chromosomes                                      would              constitute            an       initial       ap-
arm        of the        X or X-X             translocations,     are      vim-     proach         for                         investigating                    the      chemical            basis     of
tually       always        late     replicating             (42) and        usually X-inactivation                                      (50).
constitute            both      the      brightest            and     one      of the
                                                                                                                                             DISCUSSION
largest           chromosomes                 in                 T-pulse             metaphases
(Fig.          7 and    8). As           a source                   of cells,           one     might The         ability   of BmdU-dye           techniques         to detect
use    phytohemagglutinin-stimulated                                          T cells       iso-     deoxyribonucleic          acid     synthesis          in cytological
lated     from      peripheral                       blood,               established             lym-
                                                                                                     preparations         is now      amply        documented           (17, 22,
phoblasts        or long        term                 fibmoblast                  lines.     In such  28-35,        38, 42, 46,      55,    68).     A number           of these
studies,           incomplete                cell      synchronization    would                          techniques,               involving         either    fluorescent        dyes    on
produce            X chromosomes                         with      a wide    mange                       immunochemical
                                                                                                           of                              detection        of BmdU,       should      be
intensities,              but     most          of     these              should           exhibit       adaptable
                                                                                                            a                 to      flow         methodology.                The       obstacles
lower           overall         33258           Hoechst                    fluorescence.                 confronting
                                                                                                           A                   such               applications             are     significant,
class       of thymine-nich,                    late        replicating,                   structur-     but the       magnitude                   ofthe       signals       produced, e.g.,
ally       abnormal             chromosomes                         should          persist              the
                                                                                                       and       quenching                   of    33258        Hoechst             fluorescence
be        eminently              suitable                  for        purification.                    Bio-
                                                                                                         (34, 39, 43),             seems        sufficient          to warrant          efforts
chemical              studies           on          such           late        replicating               dealing
                                                                                                            X          with         potentially            confounding         factors.




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922                                                                                                LATT




      FIG     8’     fheju’              of 33258            Hoechst          to     highlight            the     fluorescence                 of an           X    X translocation               Thesecells                   were
cultured’42hr,in1thP#{232}ence                             of BrdU  and then:gi7i                                    7 hr preharveit      ulse’of’dT1O#{241}e’aain;                                       the        late-
replicating         structurally                      abnormal     X (long arrow)                               is highlighted       by bright      fluorescence.


      Previous                studies             have            shown            that          chrome- dilute           soluble           dye-nucleic                    acid       complexes                 in     iso-
somal     proteins          will                 reduce        the           amount     of 33258tonic                   buffers           as        an      index          of intracellular                      condi-
Hoechst        fluorescence                          observable                  from  deoxyri- tions                   remains                to        be established,                    a more               appre-
bonucleic         acid,      but                  such      effects             do not    show priate
                                                                                                   a                      alternative                      set of conditions                  is not            appar-
significant         BmdU                    dependence                       (43).         Moreover,        ent.
this     phenomenon                        might     ultimately                       be    turned           to The           combination                          of   BrdU,             33258          Hoechst,
advantage                    as   an       index             of   chromosome                     conden- and          light          appears              to       be   extremely             toxic         to        cells
sation         (7, 63).              Staining                  of mibonucleic       acid                  (60).
                                                                                                           by        Thus,     101         erg/mm2        of 365 nm       light                                           is
33258        Hoechst               has     been              reported      to occur    at                pH
                                                                                                          sufficient        to reduce       the survival       of BrdU-substi-
2 (27),          although,               at      pH      7 and          at   or above             physio- tuted,        33258     Hoechst-stained        cells   by   several
logic        ionic           strength,                this         effect          appears             to orders
                                                                                                              be     of magnitude.                                While              this   effect       can     be
small       (i.e.,      addition                of33258             Hoechst               to nibonu-        used  as the    basis                        of a more                sensitive        version        of
cleic     acid      solutions                     results       in             5% of the                    the Puck
                                                                                                        flue-           and    Kao                        technique                for isolating          condi-
mescence          observed                     when       the      dye         is added        to           an
                                                                                                            tional           lethal         mutants                      (56),       it      raises              doubts
identical          amount                     ofdeoxyribonucleic                acid   (39)).               about          the      ability        of               BndU-substituted             cells          to
While         the      validity                    of measurements               made       on              survive              a    fluorescence                      flow       experiment.                       How-




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                                                          BrdU-DYE                      TECHNIQUES                              AND              FLOW               FLUOROMETRY                                                            923

even,          the         precise                 limits              of the               amount                  of tolera- (51)               on chromomycin                           At        (64)).         Dye       binding               corn-
ble BrdU     and                              light        are   only   now                                 being       deter-petition           is a pheonomenon            that       should         ulti-
mined.   Moreover,                                      a number      of the                               applications       mately         be soluble       by choice         of suitable          staining
described                 e.g.   , for  detecting                              S.C.E.’s                    and     isolat- and         calibration         conditions.
ing      late               replicating           X                         chromosomes,                             will      be Electronic          energy        transfer        between          pains                                                  of
insensitive                       to biologically                             important                       but chemi- dyes           bound       to deoxyribonucleic          acid    constitutes
cally       sparse                  damage.                                                                                          a more    significant                             complication,                         primarily                 be-
       Previously                        described                     bisbenzimidazole                         deny-                cause  it is a relatively                               long              mange          phenomenon
atives            (35,            40,       49),          such           as       33342              Hoechst,                    may( e .g       . ,    Fig.        4).     Conversely,                     energy            transfer               can
prove            more                   desirable                     than             33258                Hoechst                  for
                                                                                                                                     prove              advantageous                       as      a highly               sensitive            gauge
studies              with               unfixed                cells          (1);      a few               other          deny- of proximity                              relationships                      (18,    61).    For              exam-
atives            may              possess                 a similar                    advantage                         (Table ple,  energy                          transfer          between                33258      Hoechst                and
I). It remains                             to      be       determined                             which                deniva- 7-aminoactinomycin                                    D is no               more         efficient           than
tive          will          cause                the           least       biological                           damage               that
                                                                                                                                       to              between              33258       Hoechst                    and       ethidium          bro-
live          cells,           and                the           possibility           that                        increased          mide,               even          though         the spectral                       overlap      integral
membrane                        permeability           will                            be associated                           withof the               first    dye          pain      is 5 times                  that     of the           second
increased                    staining        of cellular                                components                            other (due               primarily                to     the    high                  absorptivity                  of 7-
than    deoxyribonucleic                                    acid           is not             yet     excluded.         aminoactinomycin               D). This       implies         that      the     av-
    The    present                               report                  includes                  pilot        studies erage      separation           between         33258      Hoechst           and     7-
(Fig.           2) on a pain                          of deoxyribonucleic                        acid      bind-        aminoactinomycin                D (at a given              saturation             of
ing           dyes    ultimately                             capable                         of simultaneous            the latter)         is relatively         high,      a result         consistent
detection                   of BmdU                     substitution                         and     total      deoxy- with       the      different        binding         specificities             of the
ribonucleic                        acid   content.                         Such               two           dye         studies two dyes.                        Once          systems               are      fully     characterized,
are    subject                     to spectral                        constrainst,                          such          as the should
                                                                                                                                it                                 be          possible                to       compute            both                 the
ability              to          excite            both               species                in      the          same             cell
                                                                                                                                     amount         of energy       transfer                                  from          the    donor              and
and     to resolve        their     emission.         The     former                                                                the
                                                                                                                                   can      associated        sensitization                                       of       acceptor.                 This
often       be  accomplished              at     a single        excitation                                                 should     provide        an index        ofintemaction                                                  heteroge-
wavelength;          alternatively,        multiple       excitation                                                        neity     between          the      two.     Energy                                               transfer         may
wavelengths                             can       be used.                    Resolution                         of the two also   prove       useful      in studies         using                                            pairs         of dyes
emission                   spectra                depends                    strongly                 on         the choice as simultaneous              stains      for protein                                               and         deoxyri-
of dyes,      but      can    often     be improved                                                        by     judicious bonucleic          acid    (16). Definitive        application                                                      of en-
use     of polarizers          in front      of emission                                                         detectors. engy      transfer        to flow      systems       might        use                                                static
Additional            factors       to    be    considered                                                            include measurements            of fluorescence           for initial                                                    cali-
binding                competition                          and             intermolecular                            energy bration         followed        by    biochemical            analysis                                                          on
transfer         between         pairs of dyes.                                                                                  sorted       material           to determine           the                                    true         basis           of
    Binding          competition        between      dyes                                                            will        the
                                                                                                                                de-     sorting        criteria.
pend      strongly       on dye binding         mode   and                                                           specific-       Once        the        technical         obstacles                                         to     utilizing
ity.       Intercalating                                dyes           such            as         quinacrine                     BrdU-dye
                                                                                                                               and                   methods          for   detecting                                         deoxyribonu-
ethidium                   bromide,                       with          little         on no               G-C       binding cleic      acid   synthesis           in flow        systems        can be oven-
specificity                      (52)         compete                   directly                   (44).         This      maycome,        the   biological          applications             should    be im-
pose        a particular                        problem                       in flow              studies,             which pressive.         Flow        fluorometry              provides        a unique
are        generally                        carried                   out        under                conditions       opportunity
                                                                                                                         of                   to     bridge         cytological           studies                                                            of
saturating        dye                            binding,                     although                  the    propor- chromosome             structure,           replication           and      repair
tions      of the two                           dyes      bound                   should              be contmolla- with         biochemical           studies        of the        same     phenom-
ble by adjusting                                 the     input                 ratios,              and     the use ena.of      The     examples          suggested            regarding         S.C.E.
subsatunating                             amounts                      of        dye              might              be          ex- analysis                  (Fig.         6) and         late        replicating                   X chrome-
plored.                   Employment                        of          dyes                with             different               somes                (Fig.           7 and     8) are    but                  a few of the                  possi-
binding                modes,                   such             as     ethidium,                         which               inter- bilities.                For          example,        if an                   appropriate                   BmdU
calates              (66),          and    33258  Hoechst,                                        which             does       labelling
                                                                                                                             not                                 protocol            can        be     found,              measurements
intencalate                  (6,      53), should    minimize                                          this           effect. on intact                           cells    might     be used     to magnify      inter-
Alternatively,                             one            might              employ                 a dye            with      cellular
                                                                                                                               A-                              differences       due    to aneuploidy       on to me-
T specific                   binding             (e.g.,           33258              Hoechst)                    with            a saicism      (8)                    involving              early     or late                       replicating
G-C specific                         dye (e.g.,                  7-aminoactinomycin                                       D        chromosomes.                           These             and     additional                           applica-




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924                                                                                               LATT


tions         of BmdU-dye                      methods           for     flow            Comings
                                                                                   fluorometric          DE: Mechanisms
                                                                                                         14.                              ofchnomosome             band-
                                                                                        ing,      VIII.       Hoechst            33258-DNA              interaction.
studies             in cell          biology           should          soon       be realizable.
                                                                                        Chromosoma             52:229, 1975
                                                                                  15. Cnissman            HA,       Mullaney              PF,    Steinkamp             JA:
                      ACKNOWLEDGMENTS
                                                                                        Methods         and     applications               of flow      systems         for
    The     expert         technical         assistance            of Mn.        Mi- analysis         and sorting          of mammalian               cells,     Meth-
                                                                                        ods in Cell Biology.                 Edited        by D Prescott.            Aca-
chael       Eisenhamd              and     Ms.         Lois       Juengens           is
                                                                                        demic      9:179,     1975
greatly       appreciated,             as are        the    gifts     of bisben- 16. Crissman             HA,     Oka        MA,       Steinkamp           JA: Rapid
zimidazole           dyes       from     Dr.       H.     Loewe,         Hoechst        staining        methods          for     analysis         of deoxyribonu-
AG and of 8-methoxypsomalen                       from      the     Paul    B.          cleic    acid     and      protein         in mammalian                 cells.      J
                                                                                        Histochem          Cytochem           24:64,        1976
Elder      Company.
                                                                                  17. Dutnillaux            B, Laurent             C, Couturier            J, Lejeune
                                                                                        J: Coloration             des      chromosomes               humains           pan
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926                                                                      LATT


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