Detection of GFP expressing cells - fix or not? - (May/29/2008 )
I have expressed GFP as a reporter in my cells and want to compare number of GFP positive cells between differnet treatment. I was told it is OK to just harvest the cells and run the FACS analysis without fixation because it may destroy GFP. Should I fix my cells or not?
You don't have to fix these cells. In most cases, people need live GFP cells sorted for subcloning/stable cell line making/enriching the best expressing cells etc, so cells are not routinely fixed.
If you are permeabilizing the cells, which you are not, you need to fix the soluble GFP. People also use membrane localized GFP to avoid leakage during permeabilization.
See some links here:
I work with EGFP-tagged virusses, so I need to fix my cells before flow cytometry. The EGFP is not membrane localized or anything (checked with confocal microscopy), and I just fix with formaldehyd in PBS and do not loose anything. (checked this with unfixed versus fixed transfected cells with a deleted virus before).
In my experience fixing with PFA decreases fluorescence of the GFP, in some cases by half. However, I did notice this to be cell dependent. I use retrovirus to infect bone marrow cells, and while my retrovirus producing lines where fine after fixation, bone marrow cell GFP expression was halved after PFA treatment. So unless absolutely needed I would recomend against fixing. I left my cells in the fridge overnight with no fixation, and both cells and GFP fluorescence was still brilliant.