staining of cord blood CD34+ cells

Dennis_Young@CIS.ucsd.edu Dennis_Young at CIS.ucsd.edu
Thu Nov 21 17:56:00 EST 1996


Yash
-1) We NEVER store whole blood cold, room temperature only. Cold activates 
platelets?

-2) PE anti-CD34 reagents always seem to give higher background than FITC (Check
monocytes IgG PE vs. HPCA-2 PE).

-3) We use 100 ul blood + 20 ul Ab.

-4) We have also incubated UCB with mouse serum to lower nonspecific binding. 
Ten minutes, RT., then directly mix with antibodies.

-5) Include CD45 FITC and scatter gates to help eliminate small debris. This may
help with UCB, as not all the erythrocytes are lysed.

See The ISHAGE guidelines for CD34+ cell determination by flow cytometry, 
Sutherland, Anderson, Keeney, Nayar and Chin-Yee, Journal of Hematotherapy, June
1996.

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 at ucsd.edu   *
*                       http://www-core.ucsd.edu/flow-core.html         *
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______________________________ Reply Separator _________________________________
Subject: staining of cord blood CD34+ cells
Author:  yagrawal at messi.uku.fi at @UCSD
Date:    11/21/96 3:52 PM


Dear flowusers,
		Problem:I have been having problems with CD34 cell
staining ofwhole Cord Blood(CB). Specifically the Isotype control (IgG1-PE from
 B.D) gives non-specific staining (0.2-0.6 % of mononuclear cells). This
small amount of non-specific staining causes difficulty in interpretation 
when the CB stained with the specific HPCA-2-PE (also from BD) gives a 
positivity in  0.5 % of cells and subtracting the background gives 0% 
positivity.

Do others have the same problem, any suggestions/advice is welcome ?.

Methods used:We collect our CB in blood bags (CPDA) and the sample is 
stored at 4 C until  analyzed which is 1-4 hrs. Staining is according to 
standard methods. 50 ul of CB + 20 ul antibody is incubated for 15 min in 
cold and in dark. After this 2 ml FACSlysis solution (BD) is added, 
incubated for 10 min and centrifuged for 5 min at 400 g. The cells are 
then washed twice in PBS and analyzed immediately.


Thanking you all,
Yash Pal Agrawal
Kuopio, Finland
yagrawal at messi.uku.fi



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