This is modified from http://flowcyt.salk.edu/howto/compensation/compensation-howto.html and can be used for compensation for the overlap of AmCyan1 and destabilized EGFP, or another similar combination.
- Make samples of the same cell type including
- AmCyan1 only
- EGFP only
- the samples you're trying to test
Day 3 or 4
- Mix appropiate equal numbers of untransfected, AmCyan1-only, and EGFP-only cells. Stain with 7AAD to count only live cells.
- Run mixture on cytometer. Calibrate as usual.
- Adjust the voltage on the FL1 detector to position the negative cell population at about 100, but keep the bright FL1 population on scale.
- Adjust the voltage on the FL2 detector to position the negative cell population at about 100, but keep the bright FL2 population on scale.
- Adjust the FL2-%FL1 compensation control to bring the green-only cell population downwards until the FL2 median of this population is approximately the same as the FL2 median of the negative cells.
- Adjust the FL1-%FL2 compensation control to bring the orange-only cell population leftwards until the FL1 median of this population is approximately the same as the FL1 median of the negative cells.
For most purposes it is adequate to set compensation "by eye" as above. If you wish to be as accurate as possible however, you must now draw regions around the 3 cell populations, display statistics and make fine adjustments to the compensation controls in order to equalize the median fluorescence values. Compensation is now set correctly. If you subsequently change the voltage on either detector, or change the detection filters, you must repeat the compensation procedure!