Hi David,
Without checking the cytometer it is difficult to say. Neverthless, the
delay between lasers
is critical to the Calibur FL3/FL4 compensation. And, since all compensation is
done before log
compression, errors in PMT voltages can easily prohibit proper compensation as
well. In other
words, 100% of 20 millivolts can never be 30 millivolts. So, you need to first
rule out
instrumental calibration as the problem, then rule out reagent problems, and
then insure you
optimize the detectors such that they are operating with very similar levels of
sensitivity.
It is not uncommon, for example, to run one directly labeled tri-color as a
control
and then run another tri-color using the same settings. This is often a mistake.
The PE-Cy5 lots
can vary quite a bit, so each one may need tweaking to properly compensate with
FL2 and
FL4 due to their differing degrees of "orangeness" and "redness".
Joe
"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
This archive was generated by hypermail 2b29 : Sat Mar 10 2001 - 19:31:27 EST