Cychrome/PE staining

David Chambers davidc at flosun.salk.edu
Thu Feb 13 19:18:46 EST 1997


Can anyone shed any light on a problem we are currently having?

Here goes...

A user wants to do Cychrome, PE, and FITC with live/dead discrimination
on a blue (UV excited) dye.  I am running these on a Facstar+ with
200mW of 488 excitation (and about 50mW UV).  The problem we are seeing
is that cells stained with anti-CD3 Cychrome alone are nicely bright,
but when PE-Avidin-biotin-anti-CD5 is also present on the lymphoid cells,
a significant subpopulation of the Cychrome positive cells becomes
a lot dimmer, which produces a "tail" into the negative control region.

As far as I can tell, compensation is set up exactly right.  I am
using the "standard" FITC/PE filters in FL1 and FL2 and a 660/20 BP in
FL3 for Cychrome.  We have experimented by varying laser power, and
200mW seems optimal.

We have also varied the order of addition of the reagents when staining
and it does not matter one little bit whether Cychrome goes on first,
second or last.  Same results.

Specificity of the Cychome antibody appears good.

We are flummoxed.  Can anyone suggest a reason for this?  We really would
like to get the Cychrome staining uniformly bright!!

--David

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E-Mail: David Chambers <davidc at flosun.salk.edu>
Date: 13-Feb-97
Time: 16:18:46

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