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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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