Hi, I use three different fluoroforms in my experiment - CD4-PE , human Enolase-FITC and TRX-APC/Cy7 and try to compansate correctly especially PE & FITC. I think I do it correctly - eg stain my population with hE-FITC and then mix it with unstained population ( all done on PBMCs ) but when I am trying to do the compensation it comes to about 75% (FL2:FL1) - way too much?
I use flow cytometer with two lasers (five filters).
Did anyone have similar problem? I already did some troubleshooting and I am about to try again but will welcome any suggestions,thanks
Eddie
-augustem-
A good way to check if the compensation is accurate is to see if the geometric mean of your unstained population matches up with the stained population's mean in a different channel. Let's use an example to illustrate:
Let's say we are compensating for PE. In our compensation tube, we will have CD4+ cells, and ideally some CD4- cells. If we add CD4-PE antibody to the tube, two populations will appear along that PE axis. However, the two populations should be equally negative in every other channel Remember, we only added CD4-PE to that tube; not any other antibodies.
So, if we plot CD4-PE on the y-axis and another fluorochrome (FITC, in your case) on the x-axis, we should see two populations:
<*>CD4-PE+/Enolase-FITC-
<*>CD4-PE-/Enolase-FITC-
Next, you want to make a quadrant gate to the right of those two populations so that PE+/FITC+ is Q1 (empty), PE+/FITC- is Q2 (has events), PE-/FITC- is Q3 (has events), and PE-/FITC+ is Q4 (empty). Then you need to take the geometric means of your populated quadrants (Q2 and Q3, in this case) and compare them. That step varies a little bit depending on your software, but it's usually found under a statistics menu after selecting an individual quadrant. With a correct compensation, the means should be roughly equal and the populations should appear "lined up" along the x-axis, since they are equally negative for that signal. If the means are very different, you need to adjust the matrix values until they are close. I would aim for a difference of ~50 or less (e.g. - 1256 and 1198). Obviously, the closer the means are, the better your compensation, but as long as you're close it should be fine.
Hope that helps!
-JMoynihan-
thank you, JMoynihan! the excellent explanation? very clear even without pictures of plots! :)
-murusa-
