Hello Ray, this phenomenom seems to depend on the beads. If you amplify your FSC and SSC up to 8 as I suspect you have done according the attached dotplot then you will see these 'fragments, artefacts or whatever'. If you decrease the amplification they are gone. May be it depends on the age of the beads. I have seen the same just yesterday when I did a turbosort on BD CaliBRITE Beads (APC and PE). In the original sample there only a few of these curious events and in the reanalysis of the sorted beads you can see much more. I have attached a ppt-file of the sort and gated on these events. One can see that nearly all of them are nonfluorescent. Gate them out. I think this doesn't influence your sorter performance negatively. Regards Volker Volker Eckstein PhD Dept. Internal Medicine V University of Heidelberg Heidelberg GERMANY volker_eckstein@med.uni-heidelberg.de -----Ursprüngliche Nachricht----- Von: ray hester [mailto:rhester@jaguar1.usouthal.edu] Gesendet am: Freitag, 30. November 2001 23:37 An: Cytometry Mailing List Betreff: 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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