PE-Cy7 and compensation problems

Fischer, Randy (NIH/NIAMS) [E] fischer1 at mail.nih.gov
Sat Apr 14 21:41:48 EDT 2007


Hi Virginia,

This sounds like multiple problems.  Starting with the second problem, the PE and PE-Cy7,
this should not be an issue unless your tandem is bad.	We, and others, routinely use PE
and PE-Cy7 without any issues.	This really sounds like your PE-Cy7 has become
dissociated with the PE now being the only fluorochrome left.

With the problem of APC in your PE-Cy7, how much spillover?  I would expect very little
to none normally for two reasons-different light paths via the fiber optics from the two
different lasers and very different emission curves.  Off the top of my head this sounds
like something misaligned-have BD check it out.

By the way, I have used the BD 34 PE-Cy7 and not had problems.

Randy Fischer
NIAMS/NIH



-----Original Message-----
From: Virginia Portillo [mailto:Virginia.Portillo at manchester.ac.uk]
Sent: Thu 4/12/2007 11:48 AM
To: cyto-inbox
Subject: PE-Cy7 and compensation problems

I would like to do some multicolor experiments in a FACS Aria using the folowing
dyes/Abs:

Rh123 (FL-1)
Hst 33342
CD38 PE
CD34 PE-Cy7 tandem dye
monoclonal Ab detected by a APC- conj. sec Ab
PI

We have tryed a similar experimet in the past and had some problems using the
CD34 PE-Cy7 Ab such as:
-APC leaking on the PE-Cy7 channel
-when using PE together with PE-Cy7, could not compensate either.

All comercial Abs I use are BD so they do not differ on fluorochrome:protein
molar ratios.

I am thinking on changing my CD34 or secondary Abs but for example the emission
of CD34 Cy5.5-PerCP would overlap with APC.

I would appreciate your comments on using tandem dyes in particular PE-Cy7
conjugates and/or any suggestions about using different color combinations on
my experiment to avoid these problems.

Many thanks!

Virginia


-- 


Dr Virginia Portillo
The University of Manchester
Manchester Interdisciplinary Biocentre
131 Princess Street
Manchester
M1 7DN

Tel: 0161 306 5820
Fax: 0161 236 0409





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