Ac2 2 x300549

Apical CFTR-mediated chloride conductance was measured as previously described^{18 (link)}. Briefly, hPSC-derived cholangiocytes were grown in 96-well plates and treated for 24 h with either or combination of CFTR modulators, 3 μM VX809, 3 μM VX661 (Selleck), 3 μM R and S-VX445 (MedChemExpress), 0.5 μM AC1 (X281602), 3 μM AC2-1 (X281632), 3 μM AC2-2 (X300549) (Abbvie), or DMSO. Cells were labeled using blue membrane potential sensitive FLIPR dye dissolved in sodium and chloride-free buffer (150 mM NMDG, 150 mM gluconolactone, 10 mM HEPES, pH 7.4, 300 mOsm) at a concentration of 0.5 mg/ml. Cells were incubated for 30 min at 37 °C. The plate was read in a fluorescence plate reader (FLIPR® Tetra System or SpectraMax i3; Molecular Devices) at 37 °C. After reading baseline fluorescence, CFTR was stimulated with a combination of the cAMP agonist Forskolin (10 μM) and potentiators, 1 μM VX770 (Selleck) or 1.5 μM AP2 (X300529) (Abbvie). CFTR-mediated depolarization was detected as an increase in fluorescence, and repolarization was detected as a decrease with addition of 10 μM CFTR-specific inhibitor CFTR_Inh-172 to all wells.