Posted 24 September 2010 - 12:13 AM
Some recommendations... Don't use PI, that really interferes with GFP. You'll have all your GFP positive cells also positive for PI. Better use ToPro3, at 1:10000 dilution. It shows in the APC channel.
If you want to use PI, make some controls: GFP only (compensate for PI signal), PI only (same for GFP), unstained, both stained. Use PI at 50ng/ml final concentration (add 1:1000 directly into your cell suspension, no wash needed). Your living cells are negative for PI. No need to fix, otherwise all cells are dead = positive for PI.
Anyway, I think you will underestimate the number of dead cells. For the preparation of cells before FACS, you'll wash and trypsinize and wash again etc. So you will lose all dead cells during washes. Better think about a different approach... Or you should measure apoptosis in GFP positive cells.
I got soul, but I'm not a soldier