Some 3d plots of FSC, SSC and FL1 or FL2 might help you elucidate what the subpopulation is -----Original Message----- From: ray hester [mailto:rhester@jaguar1.usouthal.edu] Sent: 30 November 2001 22:37 To: cyto-inbox Subject: Cause of Sort 'impurity' Hi, In trying to 'advertise' one area of our flow capabilities, I ran some of the BD Calibrite red and green beads, sorted each population and then re-analyzed the sorted populations. I intended to send these to the faculty here to show how a typical 2-color cell sort might go. As you can see in the first attachment, Fig. 1 is the unsorted sample, Fig. 2, the red bead sorted population upon re-analysis, and Fig. 3, the green bead sorted population upon re-analysis. In both of the sorted populations, the highest purity I can achieve is around 96% because of some very small, non-fluorescent 'junk' that appears in the lower left quadrant - representing about 4% of the events in both sorted populations. As you can see in the second attachment, there are some events by light scatter that don't appear in the unsorted sample (Fig. 4), but do in the sorted, re-analyzed samples (one is shown in Fig. 5). At first, I thought this was from dirt/dust in the sort collection tubes - I'm sorting directly into glass tubes without any medium or buffer because I don't want to dilute the beads before re-analysis. But I rinsed all of the tubes before analysis and that didn't help. I know this isn't that big a deal - unless you're trying to sell your services - and then 4% 'contamination' might be important. Has anyone else had this happen and, if so, what was the source of the unwanted, very small, non-fluorescent particles? Thanks for any help. Ray Hester Univ. of South Alabama rhester@jaguar1.usouthal.edu
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