FL3/FL4 compensation on a Calibur

Topham, David David_Topham at URMC.Rochester.edu
Tue Jul 25 10:42:40 EST 2000

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 at urmc.rochester.edu

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