Fluorometric Assay for CFTR Function

HEK293 cells were seeded in 96-well plates (black, flat bottom; Greiner) transfected with either WT- or I1234_R1239del-CFTR constructs as previously described [32 (link),33 (link)]. The cells were treated with 0.1% Dimethyl sulfoxide (DMSO) or CFTR correctors for 24 h and Fluorometric imaging plate reader (FLIPR) buffer for 35 min at 37 °C [34 (link)]. The plate was then read in a fluorescence plate reader (excitation: 530 nm, emission: 560 nm; SpectraMax i3; Molecular Devices) at 37 °C, and after reading the baseline fluorescence for 5 min; CFTR was stimulated using forskolin (10 µM; Sigma–Aldrich, St. Louis, MO, USA) and the potentiators VX-770 (1 µM) or AP2 (1.5 µM). CFTR inhibitor (CFTRinh-172, 10 µM) was then added to inactivate CFTR. The peak changes in fluorescence to CFTR agonists were normalized relative to fluorescence immediately before agonist (forskolin) addition [35 (link)].