[Cytometry] Distinguishing Surface vs. Cytoplasmic staining

Robin Barclay robin.barclay at ed.ac.uk
Thu Jul 16 04:40:13 EDT 2009

I would expect a cell surface antigen to remain present and accessible after 
fix/perm (unless that process changes it).  It would be of interest to stain 
before fix/perm with with one fluorochrome conjugate then following fix/perm 
with another fluorochrome conjugate of the same antibody, which might 
discriminate surface and cytoplasmic expression of the same antigen??
Robin Barclay

Dr George Robin Barclay PhD
Consultant Clinical Scientist (Lead Scientist, SNBTS Group)
  & Honorary Senior Lecturer
SNBTS Cell Therapy Group
MRC Centre for Regenerative Medicine
The Chancellor's Building
University of Edinburgh

The University of Edinburgh is a charitable body, registered in
Scotland, with registration number SC005336.

----- Original Message ----- 
From: "Prashant Sharma" <prashant.sh at gmail.com>
To: <cytometry at lists.purdue.edu>
Sent: Tuesday, July 14, 2009 6:06 PM
Subject: [Cytometry] Distinguishing Surface vs. Cytoplasmic staining

> Dear List members,
> I had a rather basic question on flow and would be glad to have help: Will 
> a
> cell positive for a certain antigen on surface staining always show that
> antigen as positive after fix-and-perm cytoplasmic staining? Even if the
> antigen is not present in the cytoplasm?
> The reason I ask (this biologically unlikely question) is because it was
> suggested somewhere that one way to exclude a false positive CD41 / CD61 
> on
> blasts due to platelet adhesion would be to simply perform cytoplasmic
> staining instead of surface.
> My take on this is that since the platelets or their membranes are 
> adherent
> to the cell surface, they would (most likely) still be there after the fix 
> /
> perm steps and would therefore still give a positive signal.
> Would you say this last assumption is correct? Or to extrapolate the
> question, if we are doing cytoplasmic staining for an antigen, are we, in
> effect, doing a "cytoplasmic or surface or both" stain?
> Thanks!
> 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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