staining of cord blood CD34+ cells

Yashpal Agrawal (yagrawal@messi.uku.fi)
Thu, 21 Nov 1996 15:52:12 +0200 (WET)

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