DNA stains enable a commonly used FACS method of monitoring a compound’s effects on cell cycle progression.
Transfer cells from culture dishes to FACS tubes. Make sure you pipet vigorously to get all the lightly adherent cells. The protocol assume 0.5 million cells per sample, so scale up or down with the volumes for your samples.
If needed, stain for surface antigens.
Spin cells for 5 minutes, 500 g in a microcentrifuge. Discard supernatants afterwards.
Resuspend the cell pellet in 250 uL wash buffer (1x PBS with 5mM EDTA).
Add 125uL 100% COLD ethanol and vortex immediately. Repeat once.
Incubate at room temperature for at least 30 minutes. This cell suspension can be left at 4 C overnight instead if necessary.
Spin cells at 5 min, 500 g. Discard supernatants afterwards.
Add 250 uL propidium iodide solution (0.1 mg/ml propidium iodide in wash buffer). Place samples on ice.
The exact parameters and protocol at this point are dependent on your flow cytometer, but in general start with gating out cell debris using SSC/FSC and doublets using FL2-H/FL2-W. PI has an excitation wavelength of 488nm and maximal emission at 617nm.
Collect at around 10,000 events at low speed settings using cell count vs FL2-H as the primary indicator of cell cycle. There will be 2 primary peaks representing G0/G1 and G2 phase respectively, with the G2 phase having double the FL2 height due to having twice the amount of DNA. Cells with less FL2 height than the G1 population are sub-G1 (dying) and cells in between the two main peaks are in the S phase.