Blue membrane potential dye

HEK293 cells were grown on 96 well tissue culture plates until confluent. The blue membrane potential dye (Molecular devices) was dissolved at a concentration of 0.5 mg/ml as reported previously (Molinski et al, 2015 (link); Ahmadi et al, 2017 ). After 15–20 min of loading the dye at 37°C, 5% CO₂ and humidified air, the plate was transferred to the microplate reader (Molecular devices). Briefly, the reader was heated to 37°C and the settings for fluorescence whole well scan were turned on, with multiple points being read each well. Upon start of the experiment, baseline reads of at least 4–5 scans were made, followed by addition of drugs (2.5 µl/well). After addition of each drug again 4–5 scans were done. The experiment ended by addition of inhibitor (CFTRinh‐172 10 μM). Upon completion of experiment, the data were exported and analysed.

Protocol for FLIPR assay was adapted from Ahmadi et al. [45 (link)]. Cells were seeded to black-walled, clear-bottom 96-well plates and cultured until 100% confluency. The blue membrane potential dye (Molecular Devices) was dissolved in a chloride-free buffer (150 mM NMDG-gluconate, 3 mMKCl, 10 mM HEPES, pH 7.35, osmolarity 300 mOsm) and loaded to the cells. The plates were incubated at 37 °C with 5% CO₂ and humidified air for 30 min, then transferred to the i3 multi-well microplate reader (Molecular Devices, San Jose, CA, USA). Eleven baseline reads were made, followed by addition of 2.5 μL forskolin (Sigma, Oakville, ON, Canada) per well. After adding the drug, 31 reads were made. Then, 21 scans were made after the reaction was terminated by addition of 10 μM CFTRinh-172. For negative controls, 2.5 microliters of dimethyl sulfoxide (DMSO; Sigma, Oakville, ON, Canada) was added instead of forskolin.