Assuming your sorter is working properly I have two suggestions: 1. Your cells may be clumping slightly, your positive cells may be sticking to negative cells, such a clump be it a doublet, triplet etc would have the phenotype of a large GFP positive cell. Such clumps could dissociate when being blasted out of the sorter nozzle. To address this you could do several things, you could gate on only the small particles, suspend cells in calcium free media with a little EDTA and use BSA instead of serum in your media. A more effective way would be to stain your cells with Hoechst or some other viable DNA dye and sort the G0/G1 peak only. 2. Another possibility would perhaps be that some of your cells are dying and the GFP is leaking out. Adding PI to your post sort cells to see if the GFP negative cells are PI positive would be revealing. Good luck Simon Simon Monard FACS Lab Manager Trudeau Institute Saranac Lake NY12983 Ph 518 891 3080 X352 >>> mlh2207 <mlh2207@ksu.edu> - 4/30/02 4:45 PM >>> Hello all. We have created GFP macrophages using the pIRESneo vector with the EGFP gene inserted into the MCS. The cells were then selected using G418. Here's the problem...even with selection we have two distinct populations, one negative and one positive. We are now trying to sort the cells, but cannot eliminate the negative population. The best sort results that we have accomplished is 60% positive and 40% negative populations. Does anyone have any clues or suggestions on why this is happening and what we can do to reduce/eliminate the negative population? We had hopes of using these cells for in vivo studies. Thanks, Marcia Marcia Hart GRA Division of Biology Kansas State University 19 Ackert Hall Manhattan, KS Office:(785)532-6795 E-mail: mlh2207@ksu.edu
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