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-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@ucsd.edu *
* http://www-core.ucsd.edu/flow-core.html *
*************************************************************************
______________________________ Reply Separator _________________________________
Subject: staining of cord blood CD34+ cells
Author: yagrawal@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@messi.uku.fi
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>Subject: staining of cord blood CD34+ cells
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