Simon, I have a question about your question, not an answer I'm afraid. If I understand Volker Eckstein accurately, "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." -then this would suggest that when sorting a 'shoulder' you extend the sorting region partially into the shoulder(further right) to help exclude weaker positives, at least in order to improve purity regardless of whether those negatives upon reanalysis, were actually once weak positives. This I do in any case on the supposition that the accuracy of the sort region is never absolute due to a number of factors especially the inherent lack of resolution due to stream in air. But I thought your question was asking whether one could extrapolate the curve of the shoulders left side distribution into the negative peak, mathematically and then perform statistics on that extrapolated peak to assess purity. Perhaps I read it wrong? Gene/UCONN Health
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