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Multiplexing of DELFIA Cell Proliferation and DNA Fragmentation


									  Multiplexing of DELFIA® Cell Proliferation and DNA Fragmentation Assays with
Conventional Fluorometric Cell-Based Assays for Identification of Toxic Compounds
                                                                                                                                          Sofia Vikström, Päivi Pulli, Pertti Hurskainen, Christel Gripenberg-Lerche
                                                                                                                                                  PerkinElmer Life and Analytical Sciences, Turku, Finland

1                                  Introduction                                                                    4                                         Assay Flow Chart                                                                                          6                Oxidative stress & DNA                                     7                 Cell Proliferation & alamarBlue assays
                                                                                                                                                                                                        Figure 2. Assay flow chart
                                                                                                                                                                                                                                                                                            fragmentation                                          Cell proliferation by incorporation of BrdU, and detection of metabolic activity in cells using
    We have developed sensitive assays for Apoptosis, cell metabolism
                                                                                                                                                                                                        for multiplexing.
    and cell proliferation monitoring these events. These non-radioactive                                                                             Grow cells in                                                                                                                                                                                the growth indicator alamarBlue. The BrdU- and the alamarBlue labeling reagents were
                                                                                                                                                      96 well plates                                    All    the   assays     are                                         The cells are loaded with CM-H2-DCFDA, treated with test               added simultaneously, The homogenous alamarBlue assay is performed first and then the
    assays can be used in secondary screening in the drug discovery                                                                                                                                     performed simultaneously                                            compounds, and the oxidative stress is monitored in live-cells. CM-
                                                                                                                                                                                                        on the same samples, and                                                                                                                   cells are fixed for the end-point DELFIA® Cell Proliferation assay.
    process, for early cell toxicity testing, to identify compounds with toxic                                                                                                                          in the same wells. In                                               H2-DCFDA is a cell-permeable indicator of reactive oxygen
                                                                                                                                                   Stimulate cells with                                 addition to increasing the                                          species. The indicator remains non-fluorescent until removal of the                                   700000                                                                0,8
    properties. Combining different assays provide more information                                                                                                                                     amount of data gained form
                                                                                                                                                    test compounds                                                                                                          acetate groups by intracellular esterases and until oxidation occurs
    regarding the cytotoxic properties of compounds. The DNA                                                                                                                                            each sample, this also
                                                                                                                                                                                                                                                                            within the cell. After live-cell, kinetic measurement of oxidative                                                                                                          0,8
                                                                                                                                                                                                        saves reagents, plates, and
    Fragmentation assay is a simple, cell-based TUNEL assay for                                                                                                                                         test compounds.                                                     stress the cells can be fixed and the cell-based assay for DNA                                        500000
                                                                                                                                                                                                                                                                                                                                                                                                                                DELFIA Cell

                                                                                                                                                                                                                                                                            fragmentation performed.                                                                                                                                                    0,7
                                                                                                                   Label cells with BrdU for                      Label cells for metabolism or

                                                                                                                                                                                                                                                                                                                                                                      Eu counts
    quantitative detection of apoptosis, performed in microplate format.                                                                                                                                The labeling of the cells for                                                                                                                                             400000                                        Proliferation
                                                                                                                     proliferation assay                             oxidative stress assay             the proliferation assay, for
    The DELFIA® Cell Proliferation assay measures the effect of growth                                                                                                                                  the oxidative stress assay,                                                                                                                                                                                             alamarBlue grow th      0,7
                                                                                                                                                                                                                                                                                                                                                                                  300000                                        assay
    regulatory substances. These assays can also be combined with one                                                                                                                                   and for the metabolism
                                                                                                                                                                                                        assay is done with live                                                     7                                                                                                                                                                   0,6
    another, or combined with other conventional fluorometric assays or                                                                                                                                                                                                                                                                                                           200000
                                                                                                                                                                       Readout: Cell metabolism or      cells, without removing the
                                                                                                                                                                            oxidative stress            culture media or the test                                                   6
    applications. We have set up cell-based assays where the DELFIA®                                                                                                                                    compound.                                                                                                                                                                 100000                                                                0,6
    detection technology is combined with conventional fluorometry, and                                                                                                                                 After    performing      the                                                                                      oxidative stress                                                                                                              0,5
                                                                                                                                                                                                        measurement for the cell                                                    5                                                                                                 0
    where both assay technologies are performed on the same sample.                                                                                                                                                                                                                                                       DNA Fragmentation
                                                                                                                                                                                                                                                                                                                                                                                            30   10    3     1    0,3     0,1   0,03   0,01 0,003   0
                                                                                                                                                         Fixation of cells                              metabolism assay, the
                                                                                                                                                                                                        culture media is removed,                                                   4
                                                                                                                                                                                                        and the cells are fixed.                                                                                                                                                                           [Staurosporine (µM)]

                                                                                                                                                                                                                                                                             S /B
                                                                                                                                                                                                        The TUNEL reaction for
                                                                                                                        TUNEL reaction for DNA
                                                                                                                                                                                                        quantification   of     DNA
                                                                                                                                                                                                                                                                                    3                                                              Figure 6. CHO cells were cultured over night at 10000 cells/well in a 96-well plate. Staurosporine, 0 – 30µM, was added
                                                                                                                         Fragmentation assay                                                                                                                                                                                                       and the cells were incubated for 6 hours before addition of the BrdU- and the alamarBlue labeling reagents. The cells were
                                                                                                                                                                                                        fragmentation is performed
2        Overview of the ToxSuite Products                                                                                                                                                              directly in the wells on the
                                                                                                                                                                                                        fixed cells.
                                                                                                                                                                                                                                                                                                                                                   incubated in the presence of alamarBlue and BrdU overnight. The absorbance was measured, and the cells were fixed
                                                                                                                                                                                                                                                                                                                                                   before detection of incorporated BrdU according to the DELFIA® Cell Proliferation kit insert.

                                                                                                                                                                                                        Detection of incorporated
                                                                                                                                           Label with antibodies for            Label cells for cell
                                                                                                                                                                                                        nucleotides is done using
                                                                                                                                          time-resolved fluorometry              number control
                                                                                  Membrane damage:                                                                                                      components labeled with
       Proliferation:                                             TDA                                                                                                                                   different       lanthanide                                                  0
       DNA synthesis
                                               DELFIA EuTDA
                                               integrity TDA
                                                                                  Intracellular marker
                                                                                                                                             Measure the time-                     Measure the
                                                                                                                                                                                                        A label for total number of                                                        Staurosporine                      H2O2
                                                                                                                                                                                                                                                                                                                                                   8                 Cell Proliferation, cell metabolism, and
                                                                                                                                           resolved fluorescence                  fluorescence
                                                                                                                                                                                                        cells    in     each   well
                                                                                                                                                                                                        (SYTO24®) is also included
                                                                                                                                                                                                                                                                                                                                                                           DNA fragmentation assays
                                     Metabolism                                                                                                                                                         in the detection mix.
                                              DELFIA Proliferation
                                                                                                                                                                                                                                                                        Figure 5. Oxidative stress and DNA fragmentation in CHO cells. The         Four different assays are performed in the same well. The cell metabolism assay is an
                                                                                                                                                                                                        All    the  assays   are                                        cells were loaded with CM-H2-DCFDA and treated with different
                                              DNA synthesis                                                            Readout: DNA                      Readout: Cell           Readout: Number        measured     using   the
                                                                                                                                                                                                                                                                                                                                                   absorbance measurement, the DELFIA® cell proliferation and the DNA fragmentation
                      ADP                                                                                              Fragmentation                     Proliferation             of cells/well        multilabel plate readers                                        compounds inducing oxidative stress or apcptosis. The data is              assays are time-resolved fluorescence assays and the label to correct for unequal number
                                                                                                                                                                                                        VICTOR™ and EnVision™.                                          expressed as signal from treated- compared to untreated cells.             of cells/well is a conventional fluorescence label.
                                                                        TruPoint Caspase-3
                                                                                                                                                                                                                                                                                                                                                                10                         Proliferation (BrdU)
      General Cell death:                                Apoptosis                                                                                                                                                                                                                                                                                              9
                                                                                                                                                                                                                                                                                                                                                                                           DNA fragmentation
      DNA synthesis                                                                                                                                                                                                                                                                                                                                             8
      ATP                                                                 Apoptosis:
                                                                          Caspase 3 acitivity
                                                                          DNA fragmentation
                                                                                                                   5                                                                 Cell Proliferation & DNA fragmentation assays                                                                                                                              7                          Cell metabolism
                                                                                                                                                                                                                                                                                                                                                                6                          (alamarBlue)
                                                                                                                                                                                                                                                                                                                                                                                           amount of cells (SYTO24)

                                                                                                                                  The DELFIA® Cell Proliferation assay is based on the incorporation of BrdU into newly synthesized DNA strands of proliferating cells. Incorporated BrdU is                                                                    5
                                                                                                                                  detected using a labeled antibody. The DNA Fragmentation assay is a cell-based assay performed in 96-well micro plate format for quantitative detection of                                                                    4
                                                                                                                                  apoptosis. The labeling of the 3’-hydroxyl ends of DNA fragments provides a measure of cells undergoing apoptosis. Incorporated, labeled nucleotides can
Figure 1. Non-radioactive cytotoxicity assays are used primarily in secondary screening in the drug                                                                                                                                                                                                                                                             3
discovery process. There is a trend, for early cell toxicity testing to identify compounds with toxic properties                  be detected by labeled streptavidin. We have combined this two assays, Europium-labeled anti-BrdU antibody for the proliferation assay, and Samarium-
before lead optimization. These kinds of assays are also used in basic research, to study cell proliferation,                     labeled streptavidin for the DNA fragmentation assay.                                                                                                                                                                         2
apoptosis, etc. in for instance cancer research and immunology. The cell-based assays of the ToxSuite                                                                                                                                                                                                                                                           1
provide extensive information about the cytotoxic effects of compounds.
                                                                                                                                                                                                                                                                       10                                                                                        100                                                    1000                                               10000
                                                                                                                                          50000                                                                       200000                                                                                                One assay
                                                                                                                                                                                                                                                                       9                                                                                                                                   num ber of cells/w ell
                                                                                                                                          45000                                                                       180000                                                                                                Both assays
                                                                                                                                                                                                                                        Eu ro p iu m co u n ts

            Multiplexing of cell-based assays                                                                                             40000                                                                       160000
                                                                                                                                                                                                                                                                       8                                                                           Figure 7. CHO cells were plated at an increasing concentration. The cells were treated with 10µM STS for 6 hours. The
                                                                                                                   Sam ariu m co u n ts

                                                                                                                                                                                                                                                                                                                                                   data shows that the cell metabolism (alamarBlue) and the cell proliferation are not significantly affected by the STS
                                                                                                                                                                                                                                                                       7                                                                           treatment, while the DNA Fragmentation increases. Some of the cells are washed away due to the STS treatment, the
Toxic compounds may influence cells in many different ways,                                                                               35000                                                                       140000                                                                                                                       control for equal amount of cells/ well is used to correct for this.
through various mechanisms. All types of toxicity can not be

                                                                                                                                          30000                                                                       120000
detected by one type of cell-based assay. Some of the potential                                                                           25000                                                                       100000                                           5
drug candidates may have a toxic effects on the cell, in which
case the toxicity can be detected when running a toxicity assay
                                                                                                                                                                                                                      80000                                            4                                                                           9                                                   Conclusions
                                                                                                                                          15000                                                                       60000                                            3
simultaneously on the same sample. Time is also a critical                                                                                                                                      Proliferation                                                                                                                                          Cell-based DELFIA® assays can be multiplexed and combined with assays and
factor, some toxic effects are discovered after a short time of                                                                           10000                                                                       40000                                            2                                                                               applications using conventional fluorescence measurements.
treatment, while other require long incubations. Multiplexing                                                                                                                                                                                                           1
                                                                                                                                           5000                                                                       20000                                                                                                                            Multiplexing of DELFIA® assays saves time as well as reagents and more data is
different types of cell-based toxicity assays gives more                                                                                                                                                                                                                                                                                               gained from one sample.
                                                                                                                                               0                                                                      0                                                0
information regarding the toxic properties of a compound. Since
                                                                                                                                                                ST S                             CTRL                                                                           D N A F r ag ment at io n             Pr o lif erat io n
the DELFIA® assays are based on time-resolved fluorometry,                                                                                                                                                                                                                                                                                             All assays can be performed on the same plate and read on the same time-
                                                                                                                                                                                                                                                                                                                                                       resolved fluorescence reader.
they are readily combined with conventional fluorescence
                                                                                                                   Figure 3. The data shows proliferation and DNA fragmentation in CHO cells,                                                                      Figure 4. Proliferation and DNA fragmentation in CHO cells treated with
assays. The lanthanide chelates are well suited for multiplexing                                                   10000 cells/well, grown overnight. The cells were treated with 10µM staurosporine                                                               STS. The two assays do not significantly affect each other when                     When performing cell-based assays where cells are going through various
assays. The use of the lanthanide chelates will maximize                                                           (STS), for 6 hours to induce apoptosis. Apoptotic cells were compared to untreated                                                              performed simultaneously in the same sample wells. The data is                      treatments, it is important to include a control for equal number of cells in each
                                                                                                                   cells. The data is expressed both as Samarium counts for the DNA fragmentation                                                                  expressed as signal to background for both the proliferation- and the               well. We have used a nucleic acid stain for this. The label is not interfering with
sensitivity and minimize signal spillover for the assays.                                                                                                                                                                                                                                                                                              the time-resolved fluorescence.
                                                                                                                   assay, and as Europium counts for the proliferation assay.                                                                                      DNA fragmentation assays when performed either alone or together.

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