Fluo 4 direct calcium channel assay kit

Fluo-4 Direct calcium channel assay kit (Life Technologies, Carlsbad, CA, United States) was used, to check the effect of RP2 on human Ca²⁺ channels (Blanchard et al., 2014 (link)). Fluo-4 dye containing probenecid (5 mM) was incubated with HEK 293T cells (5 × 10⁴) for 1 h and fluorescence was measured for 15 s. At 15 s, the cells were treated separately with DMSO (mock), 50 μg/mL of verapamil, or 32 μg/mL of RP2. Then, the Ca²⁺ channel stimulator carbamylcholine chloride (50 μg/mL) was added at 60 s and the fluorescence was quantified for another 120 s on a microplate reader.

The effect of compounds (Ar1-Ar24) on human Ca²⁺ channels was evaluated using Fluo-4 Direct calcium channel assay kit (Life Technologies, Carlsbad, CA), according to the manufacturer’s instructions. As described earlier [31 (link),39 (link)], the 2X Fluo-4 Direct calcium reagent loading solution containing probenecid (5 mM) was added in equal volume to the 96-well plate containing 5 × 10⁴ HEK-293 (AddexBio, San Diego, CA) cells/well. The plates were allowed to equilibrate for 1 h at 37°C with 5% CO_2, and fluorescence (pre-stimulation) was immediately measured in a microplate reader. The cells were then treated with Ar1-Ar24 (16 μg/mL), Ca²⁺ channel inhibitor verapamil (32 μg/mL), and DMSO (mock) at 30 s. Then at 60 s, carbachol (50 μg/mL; calcium channel stimulator) was added, and the fluorescence (post-stimulation) was recorded for an additional 120 s.