How to properly set up a flow cytometer running FACS Diva software for cell cycle analysis

1. DNA Cell Cycle by Flow
Cytometry
Rich Hastings
KUMC Flow Cytometry Core Lab
3901 Rainbow Boulevard
Kansas City, KS 66160
913-588-0627
rhastings@kumc.edu
http://www.kumc.edu/flow/

2. Why do we measure cell cycle by flow?
• Fluorescent probes are available that measure
DNA in a stoichiometric manner.
• The number of probe molecules bound to
DNA is proportional to the number of DNA
molecules present.
• We are able to identify resting diploid cells,
cells synthesizing DNA and cells that are pre-
mitotic or in mitosis.

3. Propidium Iodide
• Propidium Iodide – Excitation maximum = 493
nm, Emission maximum = 632 nm
• PI is bound to DNA by intercalating between
bases with no preference for purine or
pyrimidine base pairs. PI will also bind to RNA.
• One PI molecule per 4-5 base pairs.
• PI cannot pass through intact cell membranes,
cells need to be dying or permeabilized to
allow PI staining.

4. DNA + Stain
• Follows Chemical Law of Mass Action.
• ~3 x 109 bp per cell (diploid cell in G1)
• Most intercalating fluorochromes (like PI) bind
every second bp.
• ~1.5 x 109 binding sites/cell, but a large
fraction of binding sites are inaccessible (10 to
70% depending on the fluorochrome) to the
dye.

5. DNA + Stain
• Only 1.5 – 10 x 108 sites in the cell are actually
available for binding.
• For 106 cells, there are between 1.5 – 10 x
1014 binding sites.
• Assuming that the average MW of a DNA
fluorochrome is ~300 daltons (PI = 668.4, DAPI
= 277.3), at a concentration of 100 µM there
are 6 x 1016 molecules of ligand present.
• Information from Zbignew Darzynkiewicz.

6. DAPI
• DAPI – Excitation maximum = 358 nm,
Emission maximum = 461 nm
• DAPI is bound to dsDNA in AT clusters in the
minor groove.
• Because DAPI is excited by the violet laser and
emits in the blue wavelengths, it is an
excellent counter-stain for yellow, green and
red fluorochromes.

7. Both PI and DAPI stain RNA
• Add RNase to your samples to prevent RNA
staining from contaminating your cell cycle
data.

8. Visualizing the Cell Cycle
Image from Alice Givan - Dartmouth

9. Visualizing the Cell Cycle
Images from Compucyte

10. Visualizing the Cell Cycle
• Figure from Purdue University Cytometry laboratories.
G1
M
G2
S G0
Quiescent cells
G1 (Gap1)
interval between mitosis and
initiation of DNA replication,
RNA polymerases have access to the genome,
much protein synthesis.
S
interval of time in
which the DNA is replicated
G2 (Gap2)
interval between DNA
replication and mitosis
M (Mitosis)
Dividing the replicated chromosomes.

11. Visualizing the Cell Cycle
Image from BD

12. What Good Cell Cycle Data looks like:
Fluorescence Intensity

13. Diva Desktop:
Database: Double
click to open
experiment. Right
click for most
options.
Cytometer:
parameter settings
are controlled from
here. Make sure PI
is not set to log.
Acquisition
Dashboard: Set up the
instrument with
Acquire data button,
save data with record
button. Choose
stopping gate here.
Global
worksheet:
Choose
experiment
layout here.
Choose dot plot or
histogram here.
These
buttons
are for the
various
gate types.

14. Use the FSC and SSC controls to gate
cells by size and granularity:
Use gate tool to gate population.

15. Setting PI Voltage
Make sure that PI area (A) and height
(H) are checked. PI is run in linear mode
(Log should not be checked).
Adjust PI voltage so that both the G0/G1 peak and G2/M peak are visible.
It is important to see both peaks on the histogram. The G2/M peak should be
roughly double the scale of the G0/G1 peak.
2N = 52 4N = 104

16. Doublet Discrimination
1. Create a dot-plot.
2. Use the drop down menus on
the x- and y-axis labels to choose
PI-A(Area) for the x-axis and PI-H
(Height) for the y-axis.
3. Notice the values for the G0/G1
and G2/M peaks.
4. Draw a gate around the G0/G1
and G2/M Peaks. The color of the
gate can be changed by selecting
the gate button in the gate
hierarchy (right-click on the dot
plot for the hierarchy menu).
Gate Hierarchy

17. Doublet Discrimination
5. During the staining process,
aggregates may form, we must
exclude these aggregates.
6. Notice the discrete population
under the G2/M population. These
cells are G0/G1 cells passing
through the laser intercept as
doublets. We know this because
the doublets have similar height
values as single cells but double the
area measurement. Two cells are
going through the laser intercept at
the same time.
7. Do not fret over doublet
discrimination gating. Proper gating
requires experience and the Flow
core will assist you.
G0/G1doublets

18. Doublet Discrimination
In order to understand Doublet Discrimination, we must first understand the parameters
the instrument acquires:
A parameter is a measurement of a cell property determined as the cell passes
through the laser beam. Each parameter is the output of a single photomultiplier
tube or photodiode, measuring fluorescent or scattered light.
Digital data can be measured for different pulse parameters. BD FACSDiva software
measures area, height, and width for the number of channels available for this
cytometer. Time is also a recorded parameter.
• area = sum of all pulse heights (out of 262,144 for a typical pulse)
• When measuring area, the electronics add all measurements under the
pulse, in effect increasing the resolution from 16,384 levels to close to
300,000 (for most practical applications). This is equivalent to
approximately 18 bits (218 = 262,144).

19. Doublet Discrimination
In order to understand Doublet Discrimination, we must first understand the parameters
the instrument acquires:
Area vs Height
Collecting area measurements for digital data provides greater resolution and
scalability.
• peak (or height) = maximum digitized value of the pulse (out of 16,384) To place BD
FACSDiva data on the same scale (18-bit resolution), the software multiplies all height
measurements by 16.
• width = (scaled area divided by height) x 64K
Digital data is displayed on a 262,144 scale.

20. Doublet Discrimination
Visualizing Height, Width and Area: First we must understand how each cell is
measured at the laser intercept as it transits the flow cell.
Time
VoltageIntensity
Height
Threshold
Laser intercept

21. Doublet Discrimination
Visualizing Height, Width and Area: First we must understand how each cell is
measured at the laser intercept as it transits the flow cell.
Time
VoltageIntensity
Area
For each cell or debris particle, the
electronics measure the Area and
Height values. Width is calculated
by dividing Area by height x 64k.

22. Doublet Discrimination
When measuring cell cycle by flow cytometry, there is always a chance that
doublets will complicate and skew your results.
G0
G0
G2/M
G2/M
Laser intercept
What will the voltage pulses
look like when the cells and
doublets pass through the
laser intercept?

23. Doublet Discrimination
G0
G0
G0
G2/M
VoltageIntensity
2N Height 2N + 2N Height
4N Height
2N Width 2N + 2N Width
Stained nuclei
separated by
cytoplasm
4N Width

24. Doublet Discrimination
Blue cells are the
G0/G1 doublets.
They have double
the area and width
values of single
cells but lower
height values than
the G2/M cells.

25. Cell Cycle – Wrap Up
• Good preparation = Good Data
1. RNase
2. Filter cells
• Turn on all parameters (W, A, and H) for FSC, SSC,
and PI or DAPI.
• Critical to put PI on a linear scale.
• Exclude doublets from your analysis.