Spectramax m2 fluorescence plate reader

HCT116 and PC3 cells were seeded overnight into 96-well plates in 100 μl of medium at a concentration of 5 × 10⁴ cells well⁻¹. After overnight incubation at 37°C, medium was removed and replaced with 200 μl of medium containing increasing concentrations of ETPs or vehicle control (DMSO). Plates were placed in either a normoxic incubator or a hypoxic chamber (Billups-Rothenberg; Del Mar, CA) for 18 h. Cell viability was measured by adding 20 μl CellTiter-Blue cell viability reagent (Promega; Madison, WI) to each well, after which the cells were returned to the 37°C incubator until sufficient color change. Fluorescence intensity was read at 570 nm using a SpectraMax M2 fluorescence plate reader (Molecular Devices; Sunnyvale, CA).

To assess drug efflux rate in human HCC cells, the Vybrant multidrug resistance assay kit (Molecular Probes #V13180, Eugene, OR) was used. The assay uses non-fluorescent calcein acetoxymethylester (calcein-AM) as a drug-mimic and a substrate for cancer cell efflux pumps. Calcein-AM is highly lipid soluble and permeates the cell membrane where it is converted to a fluorescent calcein by the intracellular esterases. The amount of intensely fluorescent calcein that is retained, can be measured as a measure of dye effluxed or an indication of dye retention inside the cell. The assay was performed as per the manufacturer’s protocol with some necessary optimizations. Briefly, the GH treated cells were counted and seeded at 50,000 cells/well in a black, clear bottom Costar 96-well plate (Corning #3603, Corning, NY) and then calcein-AM was added at a final concentration of 2 uM, and incubated at 37C for 2 hr. After thorough washing, fluorescence was measured at 494 (excS)/517 nm (emi) in a spectramax M2 fluorescence plate reader (Molecular Devices, Sunnyvale, CA) and SoftMax Pro v6.2.1 software. Experiments were done in quadruplicate.