staining of cord blood CD34+ cells

Yashpal Agrawal yagrawal at messi.uku.fi
Thu Nov 21 08:52:12 EST 1996


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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