3.4. MRP Inhibition Assay

The ovarian carcinoma cell lines A2780 and A2780cis were obtained from PD Dr. Dr. Hermann Lage from the Virchow hospital, Berlin, Germany. Cells were cultured in RPMI-1640 medium that was supplemented with fetal bovine serum (10%) and penicillin/streptomycin (1%) at 37 °C and under carbon dioxide atmosphere (5%). To ensure continuous MRP2 expression, the cell line A2780cis was additionally cultured with cis-platin (1 µM).

In the assay each 500,000 cells were centrifuged with 2000 UpM at 4 °C in an Eppendorf tube. The supernatant was removed, and the samples were stored on ice. Then they were resuspended in RPMI-1640 medium and test compounds and the probenecid control were added from stock solutions in DMSO (dimethyl sulfoxide) to give a final concentration of 10 µM. The samples were cultured at 37 °C and 1200 UpM in a thermomixer. Then the fluorescent CFDA was added from a PBS solution to give a final concentration of 1 µM. The samples were centrifuged again, and the supernatant was removed. Then, PBS was added, and the samples were centrifuged again. That washing procedure was repeated. Finally, the fluorescence of the resuspended cells was measured by flow cytometry using 10,000 cells and a MACSQuant Analyzer. The measurement was conducted each for three times in inhibitor treated and untreated control cells. The FAR values were calculated by division of the fluorescence of the treated to the untreated control cells.