Our lab is interested in identifying dividing human B-cell
subpopulations undergoing class switch under varying
conditions. To do this we have been trying follow BrdU
incorporation. The protocol we started out with (Caryon & Bord
J. Immuno. Meth.147(1992),pages 225-230) involves surface
staining of the cell and then fixing them over night in 1% PFA
w/0.01 Tween followed by a DNase I treatment and then an ant-
BrdUrd mAb.
We are using a mouse monoclonal conjugated to FITC from
CalTag Labs.(MD5201). Every time we have tried it we get
background in the third log regardless of whether or not the
cells have been exposed to BrdUrd. I have already tried blocking
with 10% normal mouse serum for 30 minutes prior to adding the
anti-BrdUrd. This has had no effect.
If any one has any experience or input in this I would
appreciate a nudge in the right direction
Richard Palmer
Box 1198
Mt Sinai Med. Center
New York, NY 10029
PALMER at MSVAX.MSSM.EDU
