Dear flow cytometrists: We have recently become aware of an interesting artifact regarding 4-color samples on our Calibur. The problem occurs when analyzing samples stained with a bright FL3 fluorochrome, say CD8-tricolor, and a weak FL4 stain, say CD25-biotin/SA-APC. When compensation is set properly with the single color reagents, the weakly staining CD25+/CD8+ population appears to be CD25 negative. The brighter the stain in FL3, the more negative the FL4+ cells appear. So CD25 appears OK on the CD8-negative population. We know this is not correct because when another color is used for the CD25, say CD25-PE, it appears just fine. I believe there is a time delay for the FL4 detector, and I wondered whether this factors into the compensation issue. I have also noticed that it is nearly impossible to overcompensate FL3-FL4, i.e. make the events squish against the axis. We are still experimenting with the issue to try to resolve it with different settings, but I was hoping someone could explain it to me a little more clearly. David J. Topham, Ph.D. Center for Vaccine Biology & Immunology Aab Institute for Biomedical Sciences University of Rochester Medical Center 601 Elmwood Avenue, Box 609 Rochester, NY 14642-8609 Tel. 716 273-1403 FAX 716 756-5614 E-mail: David_Topham@urmc.rochester.edu
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