OATP-mediated uptake of HMC-FMX was examined. 22Rv1 cells were pretreated with OATP inhibitors rifampicin (25 μM), CsA (20 μM) and CCK-8 (20 μM) for 3 h at 37 °C. Then, the medium was removed, and cells were treated with HMC-FMX at 8.0 μg/mL [HMC] for 3 hours at 37 °C. Medium was removed from the wells, and the cells were washed once with PBS. Next, cells were fixed with 4% PFA at RT for 10 min and stained with DAPI for 15 min at RT. The cells were imaged using a fluorescence microscope. In addition, uptake mechanism studies of HMC-FMX treated with 22Rv1 cells were analyzed by flow cytometry. Briefly, after pretreating 22Rv1 cells with different OATP inhibitors, HMC-FMX at 8.0 μg/mL [HMC] was added to the cells and incubated for 3 hours. Media was removed, and the cells were harvested by trypsinization. Cells were resuspended in FACS buffer and analyzed by LSR Fortessa flow cytometry to record the fluorescence histograms of HMC dyes.

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