non-specific binding and mitochondria staining

From: Bratislav Janjic (royal011@visto.com)
Date: Sat Apr 29 2000 - 19:33:13 EST


Hi,

How can I block non-specific binding in procedure of purification B cells from PBMNC
using negative immunoselection by immunomagnetic beads. For that purpose, I tried to use
different concentrations of human Ig (to block Fc receptors), but still, I have got only
5-6 million of B cells from 200 million PBMN cells because of non-specific binding. By
the way for this purpose I use anti-CD3, CD5, CD56, CD16, CD14 CD13 and anti-glycophorin.

My next question is about ApoAlert mitochondrial detection kit by flow-cytometry
(Clonitech company), and MitoSensor, dye that company gives in this kit for mitochondrial
staining, that is used for determination of “healthy” mitochondria and mitochondria that
are changed in the process of apoptosis. I would like to know what is the experience
of people that use this kit, because the company provide only technical support for
this kit, but not the examples of results that were got.

Sincerely,
Bratislav Janjic, B.Sc.
Department of Pathology E1005
Biomedical Science Tower
University of Pittsburgh Cancer Institute
PA 15213



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