green excitation 488 nm emission 507 nm and daunorubicin red 488 nm 575 nm fluorescence data analysis was performed using cellquest becton dickinson software evaluation of pgp expression in flow cytometric studies of the mdck mdr29 cell line was determined using a high affinity pgp monoclonal antibody that recognizes an external epitope of the protein conjugated with phycoerythrin with an f p ratio of 1 0 becton dickinson antigen saturating protein concentrations of the primary and isotype matched pe conjugated control antibody pharmingen were incubated for 30 minutes at room temperature with 250 000 cells from each sample per 50 u03bcl in pbs with 0 1 sodium azide as per manufacturer 39 s recommendations cells were washed pelleted resuspended in 500 u03bcl fixed with 1 paraformaldehyde and analyzed immediately ten thousand events were collected per sample in list mode files during laser excitation in the facscan analyzer using lysis ii software becton dickinson for each experiment identical detection settings were used for all studies data were processed using cellquest software becton dickinson all studies were conducted in triplicate with sham treated controls corresponding to each time point time course evaluations of pgp and gfp expression were conducted with noncytotoxic doses of mmc and dox as defined below cells were plated in t25 flasks falcon to insure 30 70 confluence during the time course studies 96 hours viability in selected samples throughout the time course studies was determined by addition of 7 amino actinomycin d viaprobe pharmingen pgp and gfp expression in non viable cell populations were excluded median fluorescence of.