[Cytometry] Distinguishing Surface vs. Cytoplasmic staining

Prashant Sharma prashant.sh at gmail.com
Fri Jul 17 10:46:35 EDT 2009

Thank you very much Drs. Simons , Barclay, Schrager, Prussin, and Alkan for
the very illuminating answers.

What I conclude is that,

1) cytoplasmic staining alone of fixed/permed cells will not distinguish
between surface versus cytoplasmic localization of the antigen-of-interest;

2) to be sure that the location is cytoplasmic alone, one would somehow need
to "quench" the surface epitopes completely, whether by another blocking
antibody (Drs. Schrager & Prussin), or by using the same antibody with
another fluorochrome (Dr. Barclay), and then go on to deal with the
cytoplasm; and...

3) in both the alternatives in point 2 above, the cautions about the
leftover surface epitopes and the high antibody dilutions would apply.

Dr. Alkan. I stand educated on using the IF microscope to see the cells
processed for flow, and Dr. Barclay about using the same Ab with a different
fluorochrome too. Thanks!

I had read the Betz paper in Blood, but sort of assumed that the IF sample
would need to be processed separately. This "direct visualization" really
sounds like something I could try. In fact the possibilities it opens up in
other situations too are mind boggling.

I find FCM incredible for the room for creativity and innovation that it

Best regards to all,

Prashant Sharma, MBBS, MD, DNB
Senior Resident, DM student
Hematology Department
All India Institute of Medical Sciences
Ansari Nagar, New Delhi, India.

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