Re: Bcl-2 measurement by flow cytometry

From: Andy Baker (andybaker13@hotmail.com)
Date: Mon Jan 14 2002 - 07:31:23 EST


Dear All,

I'm new to this list, but would appreciate any hints people might be able to
give me on interpretation of results I am getting.

I am measuring Bcl-2 expression in flow cytometry, looking mainly at human
peripheral leucocytes and occasional lymphoma samples.  I have also tried
the Daudi cell line, which I am told should be negative(?).  I have been
permeabilisng cells with Fix and Perm from Caltag

My real problem is that I get really inconsistent results with different
antibodies from different places.  I have tried FITC conjugated antibodies
from Caltag and Dako, and get very different results - basically everything
is positve using the Caltag reagents and weak or negative using Dako
reagents.  I've tried titrating the reagents - and both titrate very quickly
giving half log reductions in intentisty with only 1/2 dilutions. I know
they are different antibody clones - but I don't know what the "true"
results are.

I would be really grateful for any advice - especially if anyone knows a
really good control system.

Thanks in advance for any suggestions,

Andy Baker
Imperial College
London



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