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UChicago Cell Cycle

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					                                                                                   DNA cell cycle analysis is affected both by the type of
 Program No.                                                                               instrument used and the binding dye
     161                                                                                                       Ryan Duggan and David Leclerc
                                                                          The University of Chicago Flow Cytometry Facility, Chicago, IL, USA
                                                                                                               CEN Data Tables (CVs and Ratios)                               Sp2/0 Data Tables (CVs and Ratios)
Introduction
                                                                                                                                                                                 DNA Cell Cycle G1 CV
                                                                                                                 CEN Singlet Population CV
 The choice of a cell cycle dye and instrument are critical                                                                                                                                                        BD
                                                                                                                                                   BD                                      BD LSRII BC Gallios
 elements in a successful cell cycle analysis experiment. Here,                                                            BD LSRII BC Gallios                                                                 FACSCalibur
 we illustrate the predictive potential of using Chicken                                                                                       FACSCalibur
                                                                                                                                                                                 PI           4.6     4.17        3.18
 Erythrocyte Nuclei (CEN) as a tool to choose the most                                                           PI          4.89     4.74         11
                                                                                                                                                                                 DAPI         13       15         N/A
 appropriate binding dye and instrument. Also, using a variety                                                   DAPI        7.68     6.13        N/A
                                                                                                                                                                                 DC-Violet   5.32       9         N/A
 of cell cycle dyes and three distinct flow cytometry platforms, a                                               DC-Violet   2.16      4.8        N/A
                                                                                                                                                                                 DC-Orange    12       12         10.2
 BD LSRII, a Beckman Coulter Gallios and a BD FACSCalibur,                                                       DC-Orange   8.32     6.26        8.71
                                                                                                                                                                                 DC-Ruby      14       16          15
 we analyzed two parameters, the CV of the G0/G1 population                                                      DC-Ruby     8.93      8.6        13.9
                                                                                                                                                                                 DRAQ5        15       16          16
 and the G2/G1 ratio, as indicators of the instrument                                                            DRAQ5       14.2     13.9         25
 performance. Keeping those factors in mind when planning a
                                                                                                                                                                                 DNA Cell Cycle G2/G1 Calculated
 cell cycle analysis experiment is key for the acquisition of                                                    DNA Content CEN Singlet/Doublet
 quality data.                                                                                                                                                                   Ratios
                                                                                                                 Ratios (2.0 = theoretical)
                                                                                                                                                                                                                   BD
                                                                                                                                     BC                                                    BD LSRII BC Gallios
                                                                                                                                                                                                               FACSCalibur
                                                                                                                           BD LSRII Gallio BD FACSCalibur
Material and Method                                                                                                                   s
                                                                                                                                                                                 PI
                                                                                                                                                                                 DAPI
                                                                                                                                                                                             1.91
                                                                                                                                                                                             1.76
                                                                                                                                                                                                      1.96
                                                                                                                                                                                                       1.8
                                                                                                                                                                                                                  1.92
                                                                                                                                                                                                                  N/A
 CENs and the Sp2/0 cell line were stained with a variety of                                                     PI          1.98   1.99        2.01
                                                                                                                 DAPI        2.01   1.99        N/A                              DC-Violet   1.79     1.92        N/A
 DNA dyes and analyzed on two digital flow cytometers (a BD
                                                                                                                 DC-Violet     2    1.99        N/A                              DC-Orange   1.62     1.78        1.49
 LSRII and a Coulter Gallios) and an analog instrument (BD
                                                                                                                 DC-Orange   2.04     2         2.02                             DC-Ruby      1.7     1.76         1.6
 FACSCalibur). Data was acquired at a rate of 100-300
                                                                                                                 DC-Ruby     1.91   2.01        1.95                             DRAQ5       1.58      1.7        1.56
 events/second.
                                                                                                                 DRAQ5       2.02   1.99        1.86                          Sp2/0 example Pics
                                 Excitation                               Collection filter


                      BD LSRII   BC Gallios   BD FACSCalibur   BD LSRII     BC Gallios        BD FACSCalibur


   DAPI


   DyeCycle Violet
                       405 nm


                       405 nm
                                  405nm


                                  405nm
                                                   N/A


                                                   N/A
                                                                450/50


                                                                450/50
                                                                              450/50


                                                                              450/50
                                                                                                   N/A


                                                                                                   N/A
                                                                                                               CEN example Pics
   Dye Cycle Green     488nm      488nm           488nm         530/30        530/30              530/30


   Dye Cycle Orange    488nm      488nm           488nm         585/42        585/42              585/42


   Propidium iodide    488nm      488nm           488nm         585/42        585/42              585/42


   DyeCycle Ruby       633nm      633nm           633nm         660/20        660/20              660/20


   DRAQ5               633nm      633nm           633nm         660/20        660/20              660/20




 • CENs were stained with a final concentration of 5μM of the
 DyeCycle dyes and 50micrograms per milliliter of PI and DAPI                                                                                                 Conclusions
 for 30 minutes at room temperature.
                                                                                                                                                             • CENs have shown themselves to be useful tools in predicting the quality of
 • Fresh Sp2/0 cells were stained with the DyeCycle dyes and
                                                                                                                                                             the cell cycle analysis. In our hands, the DyeCycle violet and orange provided
 DRAQ5 with a final concentration of 5 μM at 37°C for 30                                                                                                     the best results. This information could potentially save a lot of time and effort
 minutes.                                                                                                                                                    when planning a DNA analysis experiment.
                                                                                                                                                             • As indicators of respectively resolution power and linearity of the detector, the
 • Sp2/0 cells were fixed with 70% iced cold ETOH and stained                                                                                                CV of the G0/G1 population and the ratio of the G2/G1 signal can provide
 with a final concentration of 5μM of the DyeCycle dyes and                                                                                                  valuable insight of the instrument performance.
 50micrograms per milliliter of PI and DAPI for 30 minutes at                                                                                                • As for instrumentation, there is certainly some linearity issues associated with
 room temperature.
                                                                                                                                                             our FACSCalibur, however both the newer digital systems, the Gallios and the
                                                                                                                                                             LSRII showed linear results.

				
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posted:12/11/2011
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