I have an established cell line that expresses GFP constitutively. Once I transfect the cell line, there shouldn't any GFP expression since the DNA payload doesn't have a fluorescence marker.
My goal is to sort the "dark" cells from the GFP+ cells, but the problem I have encountered is that the GFP fluorescence is not very not strong (weak promoter), so the negative and GFP+ populations has an overlap (histogram). I'm assuming the negative population (transfected cells) expression is real due to the autofluorescence of the cells. I guess I can gate the cells on the left side of the negative distribution ( is there no meaning to where the negative cell falls in that distribution if I leave the right side out to avoid GFP+ cells ?) Can you suggest another gating strategy? am I really capturing the entire cell diversity?
Thank you for you help in advance
Hi,
I do not get the point. Why are your transfected cells GFP negative.
However, there is a way to distinguish GFP better from autofluorescence: You take a second channel where the GFP also shows fluorescence but less (same laser but other detection window/Filter). E.g., PE, PI channel (depends on your FACS). You then plot the channels against each other. While autofluorescence in both channels correlates (since its normally multicolor), the GFP of course behaves different in the two channels. I attached a file where you may figure out what I mean. Both channels 488 laser, Channel 1: 530/30 filter (GFP channel), Channel 2 585/42 filter. You see the two seperated populations, the GFP+ population is shifted toiwards channel 1.
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