[Cytometry] Distinguishing Surface vs. Cytoplasmic staining

Prussin, Calman (NIH/NIAID) [E] CPRUSSIN at niaid.nih.gov
Thu Jul 16 12:59:24 EDT 2009


Elizabeth,

Your method assumes that surface Ab staining saturates all the surface Ag and will block the later "intracellular" Ag staining. Many of the commercial mAbs are sold at such low titers (shame on you!) that there may be a substantial fraction of surface Ag free for binding more mAb when the "intracellular" step is done. I would add a step (noted below) in which you stain with an excess of unlabeled mAb to block any extracellular Ag not bound with the labeled mAb.

Revised text:
If you want to distinguish between surface and cytoplasmic markers, stain with the surface markers first, wash thoroughly, STAIN WITH UNLABELED "SURFACE" mAb to BLOCK, wash thoroughly, resuspend in clean medium, then fix, then add cytoplasmic antibodies.

Calman

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Calman Prussin
Laboratory of Allergic Diseases
National Institute of Allergy and Infectious Diseases/ NIH
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________________________________
From: "Elizabeth R. Simons" <esimons at bu.edu>
Date: Wed, 15 Jul 2009 11:53:39 -0400
To: Prashant Sharma <prashant.sh at gmail.com>
Cc: Cytometry <cytometry at lists.purdue.edu>
Subject: Re: [Cytometry] Distinguishing Surface vs. Cytoplasmic staining

You are in effect doing both if you stain after fixation since
fixation permeabilizes the cells. If you want to distinguish between
surface and cytoplasmic markers, stain with the surface markers
first, wash thoroughly, resuspend in clean medium, then fix, then add
cytoplasmic antibodies.
By the way, platelets do not adhere to siliconized surfaces nor to
most plastics if the platelet isolation was carefully done so as not
to pre-activate ("prime") them. One way to make sure you are working
with naive platelets is to add a monoclonal antibody to one of its
adhesion proteins - it should not bind if the platelets are naive
(see on of the 1980-1990 papers from my lab).
Hope this helps.
Elizabeth R. Simons

On Jul 14, 2009, at 1:06 PM, Prashant Sharma wrote:

> 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.
> _______________________________________________
> Cytometry mailing list
> Cytometry at lists.purdue.edu
> https://lists.purdue.edu/mailman/listinfo/cytometry
>

Elizabeth R. Simons, Ph.D.
Professor of Biochemistry
Boston University School of Medicine
80 E. Concord Street
Boston, MA 02118
(617) 638-4332 phone
(617) 638-5339 FAX
esimons at bu.edu
ersimons at earthlink.net

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