Attached a scheme for the groups (flasks): one flask positive control, one group treated with the drug, one group (flask) treated with medium (live or negative control). I need to use BD apoptosis kit which has annexin and PI. Shall I divide each flask into four equal groups? (PI, AV, both,neither) ? And when I divide , do I need to divide equal number for the drug and live cells? (Eg put 100.000 in each tube with different dye of the drug and of the live cells)? .
Are you trying to set compensation for staining? If so I recommend these guidelines.
http://web.mit.edu/flowcytometry/www/compensation.pdf
I have used 10,000 cells per group as this was a common practice amongst our peers and allowed us to use smaller seeding densities + enough cells left over for other downstream experiments (PCR etc.). This also spares the flow cytometer from excessive use especially with the sheath fluid. You should trypsinize your flasks and probably just aliquot only a small fraction of them (try 100,000 as you've pointed out). That way you will have a lot of cells for downstream experiments and you can see a cell pellet when you are doing the cell prep for staining.
Dear Heba,
Are you asking if you need to do compensations for cultures with and without the drug?
If this is the case, I would use only the cells where you expect to have apoptosis/celldeath for compenseation.
You need single stained cells for each of your fluorochromes/DNA stains, and one unstained sample. For the unstained compensation control, you may choose medium treated cells if you like.
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