This is a common occurence in my hands as well. Not to start a debate about analyzing sorted populations but I would set a light scatter gate to analyze the sorted beads. The beads work well for this type of sort checking, however they are prone to degradation by the sorting process (just as cells are). I usually sort the negative beads as a reference to the sorting abilities of the sorter. Achieving 100% is not hard. I suggest you sort a positive population and the neg beads and see how it works. Jim Houston > ---------- > From: ray hester > Sent: Friday, November 30, 2001 2:36 PM > To: Cytometry Mailing List > Subject: Cause of Sort 'impurity' > > <<File: SortFL.jpg>><<File: SortLS.jpg>> > 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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