Cell cycle staining for suspension cells
Required reagents: cold PBS+1%BSA, cold Cell Cycle Buffer (0.11%citrate+0.1% Triton X100), RNAse A solution (10 mg/ml), Propidium Iodide (1 mg/ml)
∑ Culture cells to desired density levels: Its trickier to get optimal density levels for suspension cells. It always helps to do a kinetic profile of your specific conditions.
∑ Resuspend 250,000 500,000 cells per condition in PBS+1%BSA in Eppendorf tubes, in a volume of ~250 ul. Ensure cells are properly suspended by gently tapping cell pellets. Keep on ice.
∑ Add 50 ul of ice cold Cell Cycle Buffer (0.11%citrate+0.1% Triton X100) and 10 ul of RNAse A solution. Incubate cold for ~3-4 h.
∑ Add 10 ul of PI solution. Mix. Incubate cold overnight.
∑ Mix samples by gentle pipetting, load into Guava and acquire using Express Plus module.
To perform analysis on a BD or similar machine, you'll need twice the number of cells and volume.
This protocol DOES NOT require ethanol or any other fixation. This reduces the extent of clumps.
Gently tap all cell pellets, and after overnight incubation, mix samples by gentle pipetting as cells are very fragile.
If you are performing intracellular staining for specific markers, use FITC/Alexa488 secondary Abs only. Remember to have single color controls for all samples.
With intracellular staining, perform all steps up to the secondary Ab wash with PBS+1%BSA+0.3%saponin. Skip the final PBS+ wash, and instead add PBS+ to make up volume to ~300 ul. Add Cell Cycle Buffer, RNASe A and PI as described above.