Hi Simon, I have had the same problem you described and I discussed it with BD's sorter specialists. Various points have to be taken into account: Weak fluorescence can result from low antigen density, bad protein/fluorochrom ratio or insufficient amount of used antibody. Even the antigen distribution or the fluorochromes distribution (patches) when labelling cell membranes can give slightly different signals when passing the analyzing point. The fluorochrome itself (tandemconjugates or not) can give weaker or stronger signals. If a sort gate is set all cells or particles which fit these criteria will be sorted if they can be sorted (no coincidence). When passing the laser light, weak fluorecent cells may loose fluorescence by bleaching. Therefore reanalyses often show the sorted cells not fit the sort gate again. And if the desired population was close to the negative unwanted ones it is possible that during reanalyses the fluorescence of the sorted cells fall into the negatives. The only argument is loss of fluorescence during the sort. RegardsVolker Volker Eckstein PhD Dept. of Internal Medicine V Medical School of the University University of Heidelberg Heidelberg - GERMANY volker_eckstein@med.uni-heidelberg.de -----Ursprüngliche Nachricht----- Von: Simon Monard [ mailto:smonard@trudeauinstitute.org <mailto:smonard@trudeauinstitute.org> ] Gesendet am: Montag, 29. Oktober 2001 20:22 An: Cytometry Mailing List Betreff: Sort purity Hi folks. Can anyone point me to a discussion of ways to estimate the purity of a sorted population of cells. This is clearly easy when you are sorting very bright cells from negatives but more problematic when sorting cells from a "shoulder". I remember seeing a nice discussion on this subject but cannot remember where. I've been sorting some very weakly positive cells from a population, these cells post sort overlap the negative cells quite a bit. My customer seems unhappy about that. Thanks Simon Monard FACS Lab Manager Trudeau Institute Saranac Lake NY12983 Ph 518 891 3080 X352
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