David, first of all, it appears from your descriptions is that you are already over-compensated. Setting the compensation so that the FL3+ cells have the same upper FL4 fluorescence as the FL3-negative cells absolutely guarantees that you are overcompensated--and explains your observation of brighter FL3+ cells being even "more" FL4 negative. And it also explains why your CD25+ cells are not appearing positive. (See, for example, <http://www.drmr.com/compensation/Practic.html>). You must properly set compensation by lining up the medians of the FL3+ and FL3- cells in the FL4 channel (on your FL3 comp sample). NOT BY LINING UP the tops, i.e., by quadrant gates. Then, and only then, will your samples show positivity. Of course, positivity can only be judged by doing a stain that includes all of your antibodies EXCEPT the FL4 (or to use an isotype control on FL4), and setting the positive/negative gate there. DO NOT USE a complete isotype control or unstained control to set this. Finally, because of the "spread" induced by compensation (<http://www.drmr.com/compensation/ErrComp.html>), no matter how high you drive the compensation between FL3 and FL4, you won't squash FL4 completely against the axis. This should only serve to prove that you cannot visually detect when you've overcompensated! See the full compensation information at <http://www.drmr.com/compensation/> to appreciate why this is the case. mr At 11:42 AM -0400 7/25/00, Topham, David wrote: >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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