RE>PE APC crosstalk? 11/24/97
It's not necessary to have compensation if you are using FITC, PE or PerCP in
addition to the APC. However if you use a PE-Cy5 tandem you have to have
crossbeam compensation because the red diode laser will also excite the Cy5
part to the PE-Cy5 tandem giving a false signal in the FL4 parameter.
Alan Stall
PharMingen
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Date: 11/24/97 4:39 PM
To: cyto-inbox
From: f preffer
I _dont_ have an answer to simons question below... but I belive I have a
related question:
Why is it necessary to have compensation [utilizing a FACScalibur] between
FL4 and other fluor channels with 2 spatially distinct excitation sources?
thanks
frederic preffer
>
>
>
>I'm having a problem with PE and APC antibodies. I am doing four color
>analysis of peripheral blood lymphocytes, two tubes are stained with CD45RA
>Fitc, CD62L PE. CD3 PerCP and CD4 or CD8 APC. I gate on CD3+CD4+ or
>CD3+CD8+ then look at the CD45RA CD62L plot to measure the number of naive
>cells. The problem is that the CD62L staining is much dimmer when gated on
>CD3+CD8+ than with CD3+CD4- although this is essentially the same
>population of cells. This effect is most pronounced with dim CD8 APC
>antibodies, I get the same effect but less so with CD4 APC. Even harder to
>explain is the fact that I don't see this effect in all individuals. Could
>the APC be absorbing some of the PE fluorescence? Steric hindrance? Whats
>going on? Has anyone else seen this? I'm using a FACS Calibur with 4th
>color, I would just gate on the CD4 negatives but this would mean I would
>include gamma/delta cells which are quite numerous in some individuals.
>
>Any suggestions welcome
>
>Simon Monard
>Aaron Diamond Center
>New York
>
>
>
>
>
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Frederic I. Preffer Ph.D.
preffer@helix.mgh.harvard.edu
Department of Pathology- Cox5
100 Blossom St
Massachusetts General Hospital
Boston MA 02114
(617) 726-7481 {fax x2365}
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