2.3. P-gp Inhibition Assay (Calcein-AM Uptake Assay)

P-gp inhibition was tested with a calcein assay as published previously [17,18] using two different cell systems: the L-MDR1 cell line that over-expresses human P-gp and the corresponding parental cell line LLC-PK1 [19] and the P-gp over-expressing murine monocytic cell line P388/dx [20] and its parental counterpart cell line P388 as a control. LLC-PK1 and L-MDR1 cells were cultured under standard cell culture conditions with medium M199 supplemented with 10% FCS, 2 mM glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin sulphate. To maintain P-gp expression, the culture medium for L-MDR1 was supplemented with 0.64 µM vincristine. One day before using the cells for the calcein assay, both cell lines were fed with vincristine-free culture medium. P388 cells were cultured under standard cell culture conditions with RPMI 1640 medium supplemented with 10% FCS, 2 mM glutamine, 500 mM β-mercaptoethanol, 100 U/mL penicillin, and 100 µg/mL streptomycin sulphate. For maintaining P-gp expression, the culture medium for P388/dx contained 0.43 µM doxorubicin. One day before the inhibition assay, both P388 cell lines were set to the doxorubicin-free culture medium.

In brief, the assay was performed in 96-well plates with a final calcein-AM concentration of 0.5 µM for the LLC cell system and 1 µM for the P388 cell system. Cells were washed with pre-warmed HBSS supplemented with 10 mM HEPES (HHBSS) and pre-incubated with this buffer for 30 min and subsequently with the test compound for 10 min (LLC cell system) or 15 min (P388 cell system). After pre-incubation, calcein-AM was added and the cells were incubated for 60 min (LLC system) or 30 min (P388 cell system) at 37 °C on a rotary shaker (Noctua, Vienna, Austria). The corresponding time points were chosen based on the highest possible measurement range for inhibition. The uptake was stopped by transferring the plates on ice and washing the cells twice with HHBSS pre-cooled to 4 °C. Subsequently, cells were lysed in 1% Triton X-100 for 15 min. The fluorescence of the calcein generated within the cells was analysed in a Fluoroskan Ascent fluorimeter (Labsystems, Frankfurt, Germany) with 485 nm excitation and 535 nm emission filters. Each concentration of hydroxychloroquine (0.01–100 µM) was tested in octuplet and the experiment was performed in quadruplicate. 200 µM verapamil was used as a positive control. Possible quenching effects of hydroxychloroquine were ruled out because no effect on calcein fluorescence was observed in the parental cell lines.