Vybrant multidrug resistance assay kit

hPgp-mediated
efflux in Caco-2 VB cells was measured using the Vybrant Multidrug
Resistance Assay Kit (V-13180, Thermo Fisher Scientific). Each well
contained 100 μL of cells in culture medium, 50 μL of
a solution containing the TRZ-DPHM derivative, and 50 μL of
calcein-AM (total volume, 200 μL). The final concentration of
putative hPGP inhibitor in each assay ranged from 0.01 to 30 μg/mL.
Verapamil and cyclosporin A, both hPgp inhibitors, were used over
the same concentration range as that used for the TRZ-DPHM derivatives.
Caco-2 cells were used as a negative control (Figure S1). Cells were incubated at 37 °C for 15 min
after the addition of TRZ-DPHM derivatives before 1 μM calcein-AM
was added to each well (0.25 μM final concentration). After
an additional 15 min at 37 °C, cells were washed with cold tissue
culture medium (200 μL), and calcein retention was measured
by the increase in intracellular fluorescence. PBS was used for all
dilutions. All experiments were performed in triplicate.

The MXR transport activity was quantified following manufacturers’ instructions. This assay is based on the principle that cells expressing high levels of P-glycoproteins (Pgp) rapidly extrude non-fluorescent calcein AM from the cytosol across the plasma membrane, reducing accumulation of fluorescent calcein in the cytosol. Briefly, cells were incubated with 1 μM calcein AM (Vybrant Multidrug Resistance assay kit, Molecular Probes, Oregon, USA) for 30 min at 18°C in darkness. Fluorescence of the intracellular calcein was quantified at excitation 485/emission 516 nm in the same microplate fluorescence reader described before.

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.