2.5. P-gp mediated efflux assays

The presence of an active efflux pump, P-gp, was probed on U937VbR cells using the fluorescent P-gp substrate, rhodamine 123 (R123), in the absence or presence of cyclosporine A (CSA) or verapamil, specific P-gp inhibitors. Cells (5000–10,000/well) were seeded in 96-well microplates at a volume of 100 μL and incubated at 37 °C for 24 h prior to addition of R123 (5 μM) in the absence or presence of CSA (20 μM), verapamil (20 μM), PBS or DMSO vehicle control (final concentration 2.5% v/v). Cells were incubated at 37 °C for a further 40 min in the dark. Culture media was removed and the cells washed with ice-cold PBS (pH 7.4). Cells were lysed with 1% Triton-X, and an aliquot (80 μL) added to wells of a 96-well black plate. Fluorescence was then measured using an excitation wavelength of 485 nm, and emission wavelength of 530 nm using a FLUOstar Optima (BMG Labtech, Germany). Fluorescence measurements were normalised to protein content in each well as determined by a Lowry protein determination assay (BioRad, Australia).

Non-fluorescent calcein-AM is converted to a green-fluorescent calcein by intracellular esterases. The degree of inhibition of P-gp activity can be quantified by measuring the increase in intracellular calcein fluorescence. MES-SA/Dx5 cells (25,000/well) were seeded in 96-well microplates at a volume of 100 μL and incubated at 37 °C, 5% CO₂ for 24 h prior to addition of CSA (40 μM), compounds 1-5 (40 μM) or DMSO vehicle control (final concentration 2% v/v), with calcein-AM (1 μM) added immediately after. Cells were further incubated for 40 min at 37 °C before being imaged on the IncuCyte ZOOM (Essen BioScience USA) using the phase and green filters at 20 × magnification (1392 × 1040, 0.61 μm/pixel, acquisition time 400 ms). All treatments were in triplicate and 4 images per well were acquired (total n = 12). The approximate number of cells scanned was 300-400 cells per image. IncuCyte ZOOM 2015A software was used to determine the fraction of calcein positive cells (defined as cells with a green pixel intensity greater than that observed for cells not incubated with calcein (i.e. green pixel intensity >10 GCU)) by dividing the average green fluorescence confluence by the average cell confluence.