I added PI to the cell line that I am interested to exclude dead cells. Then run the experiment in FACS canto. But GFP is sowing the peak at 10 to the power of 5 in FITC channel for all the samples. I dont know what to do. Maybe it's a problem with compensation.
Seems like a compensation problem or voltage/gain may be set too high. This may also happen when the lasers are not calibrated. You could try to use settings from an older experiment. PI should give signal in FL2 and FL3.
thank u korcan
but this time i want to do experiment without PI . i will collect the cells wash it and run flow. then will see.i just want to see GFP peak.
GFP and PI emission are far enough to get minimal interference.
If your FACS is functioning properly (worth checking) setting compensation parameters should be easy.
Run samples of: Negative cells; GFP only; PI only. And follow compensation instructions from manual.
As suggested by others it might be worth checking the settings of your machine but I think an easy thing to try is to lower the voltage for the FITC channel. You have to do this before you calculate the compensation though.
- Badges
- Science method