Dear Subramanian: The BD antibody (and another direct conjugate marketed by Coulter, I think 15D3) does not work on cells that express low levels of the Pgp antigen. These worked fine when used as 2 step reagents, but seemed to have lost something upon conjugation and commercialization. This is why we used MRK-16 - it works the best even on lower levels (see "Taylor BJ, Olson DP, Ivy SP. Detection of P-glycoprotein in cell lines and leukemic blasts: failure of select monoclonal antibodies to detect clinically significant Pgp levels in primary cells. Leuk Res. 2001; 25(12): 1127-35"). To detect Pgp expression and function simultaneously, one could do the functional assay, then stain for the protein ON ICE to prevent further efflux. We chose not to do this, because compensation between the colors we were using at the time (rhodamine 123 and PE) could easily distort the very low levels of clinical Pgp staining. I suppose that if the fluorochromes used had different enough emeission spectra, though, this would be feasible (for instance, rhodamine 123 or calcein used with MRK-16 and goat anti mouse APC "should" work fine).
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