DNA cell cycle analysis is affected both by the type of instrument

DNA cell cycle analysis is affected both by the type of instrument used and the binding dye

Program No. 161

Ryan Duggan and David Leclerc The University of Chicago Flow Cytometry Facility, Chicago, IL, USA CEN Data Tables (CVs and Ratios)

Introduction The choice of a cell cycle dye and instrument are critical elements in a successful cell cycle analysis experiment. Here, we illustrate the predictive potential of using Chicken Erythrocyte Nuclei (CEN) as a tool to choose the most appropriate binding dye and instrument. Also, using a variety of cell cycle dyes and three distinct flow cytometry platforms, a BD LSRII, a Beckman Coulter Gallios and a BD FACSCalibur, we analyzed two parameters, the CV of the G0/G1 population and the G2/G1 ratio, as indicators of the instrument performance. Keeping those factors in mind when planning a cell cycle analysis experiment is key for the acquisition of quality data.

Material and Method CENs and the Sp2/0 cell line were stained with a variety of DNA dyes and analyzed on two digital flow cytometers (a BD LSRII and a Coulter Gallios) and an analog instrument (BD FACSCalibur). Data was acquired at a rate of 100-300 events/second. Excitation

CEN Singlet Population CV BD BD LSRII BC Gallios FACSCalibur 4.89 4.74 11 PI 7.68 6.13 N/A DAPI 2.16 4.8 N/A DC-Violet 8.32 6.26 8.71 DC-Orange 8.93 8.6 13.9 DC-Ruby 14.2 13.9 25 DRAQ5

DNA Content CEN Singlet/Doublet Ratios (2.0 = theoretical) BC BD LSRII Gallio BD FACSCalibur s 1.98 1.99 2.01 PI 2.01 1.99 N/A DAPI 2 1.99 N/A DC-Violet 2.04 2 2.02 DC-Orange 1.91 2.01 1.95 DC-Ruby 2.02 1.99 1.86 DRAQ5

Sp2/0 Data Tables (CVs and Ratios) DNA Cell Cycle G1 CV BD BD LSRII BC Gallios FACSCalibur 4.6 4.17 3.18 PI 13 15 N/A DAPI 5.32 9 N/A DC-Violet 12 12 10.2 DC-Orange 14 16 15 DC-Ruby 15 16 16 DRAQ5

DNA Cell Cycle G2/G1 Calculated Ratios BD BD LSRII BC Gallios FACSCalibur 1.91 1.96 1.92 PI 1.76 1.8 N/A DAPI 1.79 1.92 N/A DC-Violet 1.62 1.78 1.49 DC-Orange 1.7 1.76 1.6 DC-Ruby 1.58 1.7 1.56 DRAQ5

Sp2/0 example Pics

Collection filter

BD LSRII

BC Gallios

BD FACSCalibur

BD LSRII

BC Gallios

BD FACSCalibur

DAPI

405 nm

405nm

N/A

450/50

450/50

N/A

DyeCycle Violet

405 nm

405nm

N/A

450/50

450/50

N/A

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 for 30 minutes at room temperature. • Fresh Sp2/0 cells were stained with the DyeCycle dyes and DRAQ5 with a final concentration of 5 μM at 37°C for 30 minutes. • Sp2/0 cells were fixed with 70% iced cold ETOH and stained with a final concentration of 5μM of the DyeCycle dyes and 50micrograms per milliliter of PI and DAPI for 30 minutes at room temperature.

CEN example Pics

Conclusions • CENs have shown themselves to be useful tools in predicting the quality of the cell cycle analysis. In our hands, the DyeCycle violet and orange provided the best results. This information could potentially save a lot of time and effort when planning a DNA analysis experiment. • As indicators of respectively resolution power and linearity of the detector, the CV of the G0/G1 population and the ratio of the G2/G1 signal can provide valuable insight of the instrument performance. • As for instrumentation, there is certainly some linearity issues associated with our FACSCalibur, however both the newer digital systems, the Gallios and the LSRII showed linear results.