Three Colour Compensation

Alice.L.Givan@Dartmouth.EDU
23 Sep 92 16:01:03 EDT

Has anyone noticed difficulty with compensation -- using three-colour
staining with FITC, PE, and a PE-Tri-colour conjugate. Using a BD FACScan,
I find that when we get the crosstalk of PE into FL3 correctly compensated,
we then are stuck with over-compensation of the FITC signal into FL3.
In other words, based on the amount of FL2 signal recorded from FITC and
PE and the amount of spill over into the FL3 channel from each of these
fluorochromes, they would each require different amounts of compensation
into the far red channel. How can I set the cytometer correctly for three
colour work with bright stains?
If you want to confirm this problem, set a screen so as to look at
FL1 vs FL3 and FL2 vs FL3 at the same time. Then run through a mix of
fitc-bright and PE-bright beads. Use the FL3-FL2 compensation control to
correctly set the FL2 signal in the FL2vs FL3 dot plot. Watch what happens
to the FITC beads in the FL1 vs FL3 dot plot.

Thanks for any suggestions.
Alice Givan