I am working with a group that has transfected a cell line with a GFP mutant GFPS65T. We are essentially following the protocol published by Lybarger et al in Cytometry 25:211-220 (1996). Our transfection, and fluorescence detection are comparable to what is described. I then sort out the top 15% or so of the brightest FITC labeled cells, out of the right arm of the gaussian (no second peak is distinct for the positives, but rather what we see is a continuum of increasingly brighter cells). Everything looks great for the sort (I check a sample under the fluorescent scope, number of sorted events are accurate, etc.) until I run a portion of the sorted sample back through to check for purity. I get about an 80:20 ratio of positive to negative cells, which fall out in 2 distinct peaks, scatter is still perfectly intact. I am convinced this is not an instrumentation problem, since I do not see this with any other sorted samples (which are mostly stained with surface AB's). Cells are gated for scatter to exclude debris, and a cursor is set for the positive FITC events. I am running on an Elite ESP at about 400 cells/sec, coincidence = complete abort, and negative logic. Does anyone know how fast this protein bleaches? Is it possible that I am repeatedly killing off the dim 20% of the sorted GFP? (This phenomena occurs even after a second sort of the previously enriched cell pop, at the same % frequency.) Is there a better way to set up the sort logic? I would like to improve the purity, or at least have an explanation as to what is going on. Thank for the input! Viki Mosiman RHL Flow Cytometry Core Research Facility Northwestern University Chicago IL
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