[Cytometry] Distinguishing Surface vs. Cytoplasmic staining

Jeffrey Schrager geofredo at gmail.com
Thu Jul 16 08:11:40 EDT 2009


It seems to me that if cells are positive for CD41/CD61 before fix/perm
(i.e., surface positive) and if another group that is stained after fix/perm
are also positive for these markers, how would that prove that these
antigens are truly inside the cells because the antigen can still bind to
the surface.  I suppose that if the surface is first blocked, then the cells
are fixed/permed, then stained, that may truly show if the antigen is
cytoplasmic.
Jeff

Jeffrey A. Schrager, M.D.
Genzyme Genetics


On Thu, Jul 16, 2009 at 4:40 AM, Robin Barclay <robin.barclay at ed.ac.uk>wrote:

> 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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> >
> >
>
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