[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
allows.

Best regards to all,

Prashant
_____________________________
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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