Re: Cd34
Rehse, Mark (REHSEMA@cellpro.cellpro.com)
Wed, 25 Oct 1995 14:40:12 -0700
Ed,
In response to your query on CD45 gating for CD34 analysis here's what we
do at CellPro. Since our source material is generally GCSF-mobilized
apheresis product no lysis step is needed to eliminate RBCs, however,
some residual must be gated out along with platelets. To get as objective
an evaluation of the %CD34 as possible we stain 5-10 x 10^5 cells in 100
ul buffer of PBS+100 ug/mL Human Ig with CD45FITC/CD34PE reagents. We
prefer Class III CD34 receptor recognizing antibody such as HPCA-2 PE or
581 (Immunotech claims to have this directly conjugated to PE but not
available yet) or Diaclone BF23 (same status). QBend-10 (Class II) does
not provide as good a signal separation as HPCA-2 presumably because of
the more heavily glycosylated binding sites.
We incubate for 1 hr at 4C, wash 2x in the buffer and resuspend in PI- or
7AAD-containing buffer. On the FACS acquire 30K events. Gate on the
bright and dim CD45 cells but remember that platelets will pick up CD45
so either discriminate them out or gate them out with the FSC vs CD45
FITC gate. We have definitely moved away from FSC vs SSC light scatter
gating and replaced it with a combination of FSC and CD45 gating. Much
more objective
Sincerely,
Mark Rehse
Rehsema@CellPro.CellPro.com
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