Quantifying Multidrug Resistance Protein Expression

The analysis was performed according to previously published methods [13 (link)]. Confluent cells were seeded in 24-well plates (2 × 10⁴ cells/well), allowed to adhere overnight, then treated as described above. After treatment, cells were fixed in pure methanol and washed with 4% bovine serum albumin (BSA) and PBS + Tween 20 (PBS-T) for 1 h. Then, the primary antibodies against MDR1/ABCB1 (mAB #13342, Cell Signaling Technology, Danvers, MA, USA; dilution factor 1:400), MRP1 (sc-18835, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA; dilution factor 1:500), and MRP2 (bs-1092R, Bioss Inc., Woburn, MA, USA; dilution factor 1:200), followed by staining with specific secondary antibodies (dilution factor 1:800) and nucleic acid dye (Hoechst 33258, 1 µg/mL), were added. The expression and localization of Pgp, MRP1, and MRP2 pumps into cells were analyzed by using a Cytation 1 Cell Imaging Multimode Reader (Biotech, USA). Fluorescence intensity was determined by Gen5™ Microplate Reader and Imager Software 3.11, and normalized with respect to cell number.