How to sort real GFP positive cells? - autofluorescence VS GFP (Apr/14/2005 )
hello everyone, i transfected my cells with some promoter driven GFP and there are a few GFP positive cells. i would like to sort these GFP positive cells but the problem is my cell line has a few cells with autofluorescence, which is green just like GFP and it's spot-like. the real GFP positive cells have the fluorescence evenly spreading all the cell body. could anyone give me some advice on how to sort the real GFP positive cells without the contamination of autofluorescent cells? thanks in advance.
Are they the same size? Perhaps the cell sorter can use size as a differential? Can you run a negative control cell line (transfected with empty vector or something) and gate out the ones that give you "background"?
Have you viewed your cells by flow cytometry?
Do the GFP positive cells shift to the right, above non-transfected cells?
If yes, then you can sort very easily.
If the shift is minor, just take the top 2-5% expressing cells (therefore you may need a lot more cells to begin with).
If you get a much larger shift, where > 50% above background, take the top 30-50%.
The main thing for sorting is that by flow, you can see the GFP +ve cells as a distinct population even if it is a very low percent.