2.5. Flow Cytometric OATP1B1 and OATP1B3 Inhibition Assay

As cell models for human OATP1B1 and OATP1B3, HEK293, a human embryonic kidney cell line, stably transfected with OATP1B1 (HEK-OATP1B1), OATP1B3 (HEK-OATP1B3), or the empty control vector (HEK293-VC G418) were used [23,24]. Cells were cultured under standard cell culture conditions with DMEM supplemented with 10% FCS, 2 mM glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin sulphate, and 800 µg/mL G418. Inhibition of the OATPs was quantified by flow cytometry assessing the uptake of 8-FcA into HEK293 cells over-expressing the respective transporter after 10 min incubation and normalised to the control cell line as described previously [25]. Intracellular fluorescence was quantified in a MACSQuant 10 Analyzer (Milteny, Bergisch-Gladbach, Germany) with the 488 nm laser for excitation and the 525 nm bandfilter for emission (FITC channel). Excluding debris, living cells were gated in the forward/sideward scatter by using the unstained sample. For each concentration (0.1–100 µM), 30,000 cells were measured and each experiment was performed in triplicate. Rifampicin (20 µM), a well-known inhibitor of OATP1B1 and OATP1B3, was used as a positive control. Possible quenching effects of hydroxychloroquine were ruled out because no effect on 8-FcA fluorescence was observed in the parental cell lines.