Determination of P-gp–mediated efflux of four fluorescent probes. Each probe was incubated with the parental K562 or MDR1-transfected cell line at 37°C, and mean fluorescence intensity was measured as a function of time. Verapamil (50 μg/mL) was added to each sample at the time of washing to prevent efflux before fluorescence measurement. When included as a modulator of efflux, either cyclosporin A (20 μg/mL) or verapamil (50 μg/mL) was present during the incubation period, as well as during analysis. Symbols in each panel represent parental K562 cells (□, ▪) or MDR1 transfected K-562 cells (○, •); open symbols represent uptake in the absence of modulators, while closed symbols indicate that either cyclosporin A (B and C) or verapamil (A and D) was present during probe loading.