[Cytometry] Distinguishing Surface vs. Cytoplasmic staining

Elizabeth R. Simons esimons at bu.edu
Wed Jul 15 11:53:39 EDT 2009


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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