I'm doing a differentiation experiment with flow analysis, comparing two cell lines: one is the wt cell line, and the other is a stable line transduced with our GOI + GFP marker.
The GFP is just a marker and is not important for my experiment. Can I leave the GFP uncompensated? The panels I'm using are:
(1st panel) PerCP-Cy5.5(Blue A), PE-Cy5(Y/G C), Alexa700(Red B), and sytox orange(Y/G E).
(2nd panel) APC(Red C), APC-Cy7(Red A), and sytox orange(Y/G E).
I checked the spectra and it doesn't look like GFP will overlap with the other fluors? Also, I'm worried that I can't really compare the GFP- cell line with the GFP+ cell line if I compensate for GFP; it might overcompensate the GFP- cells.
Hi Kelly,
Its true that there shouldn't be much spillover from GFP into your other fluorescence channels, but I would still include it in the compensation nonetheless (i.e., include a 'GFP only' compensation control). And you absolutely can compare the GFP+ and GFP- cell lines, as long as you stain them with the same panel and apply the same compensation matrix to the other fluorescent parameters. If you do this, the amount of compensation for GFP spillover into other channels will be the same, regardless of whether or not the cells express GFP.
Hope this helps,
Max
Hi Kelly,
If you expect to see fluorescent shifts in the colors you are interested in I would suggest compensating. Although the GFP may not contribute to the other fluorophores in this experiment, but it may in the future. It's a good habit to get into because it eliminates one more confounder. Finally, if you're using FlowJo you can remove the compensation after the fact and look at the differences. Here is a article (of sorts) on compensation.
Good luck,
NM
Dear Kelly McGlynn,
This is really a very hard question!. To begin with, you need to describe what wavelengths and from what light sources they originate. Then you need to describe the detection side, which filters/dichroics you are using, on what type of detectors. The problem with GFP, YFP, CFP, RFP is that they have very wide emission spectra, which make it almost impossible to get rid of their signals. It can be done, but it requires a proper definition of your experimental configuration of the FCM you are using. The terminology you use for describing the different panels does not ring a bell. I am used to see filter parameters in nm with their bandwidths. But I may be a bit old school;)
kind regards, Harrie Verhoeven.
Thank you everyone for your answers! Your comments are really helpful! I'll compensate for the GFP.
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