Transcript 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.