I have gfp expressing cells. Almost all of the cells are expressing GFP. But somehow the intensity of flourescence is very low and therefore it cannot be detected by FACS. Can someone tell me other way to prove quantitatively that all my cells are expressing gfp? That will be great. Thanks.
It sounds strange. GFP+ cells are detected by FACS even if fluorescence is relatively weak . Are you using non-fluorescent cells of the same type to adjust instrument parameters? You should see these cells in the lower left corner of your fluorescence plot (autofluorescence control). If you have PFA fixed cells, non fluorescent controls should be treated in parallel. Then run your GFP+ cells. Otherwise may be cells are damaged.
I think your transfection/infection is not good. Try infecting cells again with GFP plasmid again and do the sorting with FACS. Even though cells are expressing low GFP, you should be able to sort cells using FACS.
Thank you very much for the answer. Actually, I didn't fix the cells for I wanted to analyse the viable cells. And certainly I included non GFP expressing cells as a control to set the parameter. When I see under the microscope almost all of my cells are expresing gfp. But flow cytometry would detect just around 30%. And I am not even sure if that 30% is my signal cause along with the gfp channel, I am also using another channel (to observe the viabilty) which can cause spillover to the GFP channel .
Hi, I am agree with everybody that you should be able to detect them by FACS. I know what is happening!
If you are seeing all your cells are GFP positive under microscope but they are GFP negative by FACS. It means you are looking at non-specific fluorescence of GFP which is in all of your cells. If the transfection was real, it should not be visible in all of your cells under microscope. You should have seen only in some specific cells unless you have a very good negative control. That is the reason that FACS is confirming that your cells are not transfected efficiently.If you have a negative control next to your transfected cell and now look at the GFP expression under microscope, and use the same exposure time for both your positive and negative cells to detect GFP fluorescence, then you could certainly tell that your cells are efficiently transfected by GFP.
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