In our lab, we frequently pan cells that are transiently expressing the B
cell marker CD20 (used in this case merely as a co-transfection marker).
However, our results have been quite variable, and not all the
variability seems to be do to efficiency of expression of the CD20
transient (we still can pick out a CD20+ population by FACS). I'm
interested in knowing what protocols people use for 1) preparing plates
or flasks for panning (conditions used for coating with antibody) 2) the
cell labeling step (1o Ab incubation--length? conc of cells? conc of
Ab? temperature?) and 3) the panning itself (length of incubation?
medium/buffer used? temperature?). Thanks very much.
--
Bill Meikrantz
meikrant at fas.harvard.edu