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<I've been using Pharmingen FITC labelled anti-human CD19 mAbs to label B
cells from human peripheral blood, as part of a two colour assay assessing
B7.1 and B7.2 (both labelled with PE) levels on B cells. I am staining 75
microliters of whole EDTA anticoagulated blood with 15 microlilters of
antibody. I am unhappy with the sharpness, FL1 intensity, and % gated of
single positive cells (i.e FITC-CD19 only) I am obtaining. When gating on
lymphocytes = G1 on a SSC v.s. FSC plot the CD19 positive cells (i.e. LR
quadrant of FL2 v.s. FL1 dot plot of G1) from a FITC-CD19 only sample on
average make up only 7-8% of gated cells. What might be a better marker for
B cells? CD22?>
In our experience the intensity of staining with CD19 varies greatly from clone
to clone - our routine CD19 is Coulter's B4 which has a much lower fluoresence
than their B4-Lytic.
In addition we have found that the whole blood lysis procedure also affects the
final separation of pos and neg.
You do not mention your lysis procedure.
Coulter's Q prep system with the above B4, for example, gives much better
results if the tubes are allowed to stand for at least 15 mins before analysis.
CD 20 and CD22 are viable alternatives but again will vary from clone to clone
and with methods.
Wal Sharp
SQU
Oman
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