Robbins Scientific Corp
Sunnyvale CA USA
800-752-858
or 408-734-5800
Dennis
*************************************************************************
* Dennis J. Young Voice : (619) 822-0407 *
* Flow Cytometry Core Facility FAX : (619) 822-0412 *
* University of California, San Diego USA e-mail: djyoung@ucsd.edu *
* http://www-core.ucsd.edu/flow-core.html *
*************************************************************************
______________________________ Reply Separator _________________________________
Subject: Cord blood
Author: williams@ichr.uwa.edu.au at @UCSD
Date: 7/31/97 1:10 PM
Hi Flow people
I'm working on cord blood phenotyping, interested in rare leukocyte subsets.
As those who work on these preps will know, there is a problem with RBC and
erythroid contamination, variable but up to 80% of ficoll separated PBMC.
They don't seem to lyse easily using hypotonic methods, and as some are
nucleated, I wouldn't expect this to be an ideal approach.
Gating on FS/SS can partly improve percentage of CD45+ cells but not much.
We really want to clean the preps up, and I have been thinking of using
complement lysis of the RBC, perhaps with an antibody to Glycophorin A.
There are antibodies of different isotypes available, including IgM, I
presume this might be the best to start with for complement lysis. I
believe GlyA has a complement regulatory function, I don't know if this
will be a problem.
Does anybody have any hints on this, either the lysis method, or is there a
simpler way of removing the erythroid cells that I have missed out on?
Immunomagnetic separations can get a bit expensive, and positive selection
of CD45+ cells will cause problems with further analysis. I presume there
must be a lot of people doing this sort of thing, because of the CD34
interest.
Thanks.
______________________________________________
Dr William Smith
Institute for Child Health Research
(Company Limited by Guarantee ACN 009 278 755)
Subiaco, Western Australia, 6008
PO Box 855 West Perth WA 6872
Ph 61 8 9340 8792/8388, Fax 61 8 9388 3414
email williams@ichr.uwa.edu.au
______________________________________________
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>Date: Thu, 31 Jul 1997 13:10:53 +0800
>To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu>
>From: Dr William Smith <williams@ichr.uwa.edu.au>
>Subject: Cord blood