Hi Marcia, one problem of your sort maybe is autofluorescence in your cells. A second is clumping, that means you have GFP positive and negative cells sticking together. We had the same problem and now sort only the smallest part (foreward versus sidescatter) of a cell population, which have to be still GFP-postive. Taking only the small cells, you exclude 1) cell clumps and 2) big cells with high autofluorescence. We thereafter get much higher efficiencies (more than 90% positives). Hope this helps Andreas Date sent: Tue, 30 Apr 2002 15:45:17 -0500 From: mlh2207 <mlh2207@ksu.edu> Subject: sorting GFP macrophages To: cyto-inbox > > 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 > PD Dr. Andreas Simm Universitaet Halle Wittenberg Klinik fuer Herz- und Thoraxchirurgie Ernst-Grube Str. 40 D-06120 Halle Tel.: +49 (0) 345 557 2647 552 2878 FAX: +49 (0) 345 557 2782 552 2890
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