*4.3. Drug Transport Assay*

Transiently-transfected HEK293T cells (5 × 10<sup>5</sup> ) were incubated at 4 ◦C for 20 min with saturating levels (0.5 µg) of anti-ABCB1 antibody (4E3; Abcam, Cambridge, UK) in transport buffer (DMEM, high glucose (4.5 g/L), minus phenol red, supplemented with 1% FBS. The cells were pelleted at 500 G for 2 min and the supernatant discarded. The cells were washed once in transport buffer and resuspended in warm (37 ◦C) transport buffer containing saturating levels (2.5 µg) of goat anti-mouse secondary antibody, conjugated to recombinant phycoerythrin (RPE; Dako, Santa Clara, CA, USA) and one of three greenfluorescent drugs at a final concentration, unless otherwise stated, of 0.4 µM OREGON-GREEN Taxol bis-acetate (OG-taxol; Invitrogen, Waltham, MA, USA), 0.5 µM Calcein-AM (ThermoFisher Sci, Waltham, MA, USA), or 0.8 µM BODIPY-verapamil (Invitrogen, Waltham, MA, USA). The cells were incubated at 37 ◦C for 20 min before pelleting and washing as before. The cells were then resuspended in a 400 µL transport buffer and kept on ice until flow cytometry. Single fluorophore samples were also included to control for spectral spillover during flow cytometry. Two-color flow cytometry was performed on a LSRII (Becton Dickinson, Franklin Lakes, NJ, USA). Fluorescence data from 10,000 cells of normal size and granularity were acquired in CellQuest software (Becton Dickinson, Franklin Lakes, NJ, USA) and analyzed in Flowjo (Becton Dickinson, Franklin Lakes, NJ, USA).
