Fluo 4 direct assay kit

For the Fluo-4 Direct™ Assay Kit (Invitrogen, Waltham, MA, USA), 100 µL of a 5 $\times$ 105 cells/mL suspension of CHO-hM₁ cells were seeded in black clear bottom 96-well plates (Corning^®, Corning, NY, USA). After settling of the cells for 24 h, the kit was used according to the manufacturer’s protocol. In detail, the medium was removed, and 50 µL of Hanks’ Balanced Salt Solution (HBSS) was added, followed by 50 µL of the Fluo-4 buffer solution (including probenecid). The 96-well plates were incubated for 60 min at 37 °C in the dark. For the agonist assay, 100 µL of a double-concentrated dilution series of carbachol (positive control) and compounds 1–12 were added with the end concentration of 100, 10, 1, 0.1, 0.01 and 0 µM. The relative fluorescence was measured with an excitation wavelength of 494 nm and an emission wavelength of 516 nm. For the antagonist assay, 50 µL of a 4-fold concentrated dilution series of scopolamine hydrochloride (positive control) and compounds 1–12 were added. Subsequently, an 80 µM stock solution of carbachol was added to all wells, and the relative fluorescence was measured with an excitation wavelength of 494 nm and an emission wavelength of 516 nm. Stock solutions of the compounds were in DMSO with a final concentration not exceeding 1% of DMSO.

For the Fluo-4 Direct™ assay kit (Invitrogen, Waltham, MA, USA), 100 µL of a 5 × 10⁵ cells/mL suspension of CHO-hM₁ cells was seeded in black clear-bottom 96-well plates (Corning^®, Corning, NY, USA). After settling of the cells for 24 h, the kit was used according to the manufacturer’s protocol. In detail, the medium was removed, and 50 µL of Hanks’ balanced salt solution (HBSS) was added, followed by 50 µL of the Fluo-4 buffer solution (including probenecid). The 96-well plates were incubated for 60 min at 37 °C in the dark. For the agonist assay, 100 µL of a double-concentrated dilution series of carbachol (positive control) and compounds was added with the end concentration of 100, 10, 1, 0.1, 0.01 and 0 µM. The relative fluorescence was measured with an excitation wavelength of 494 nm and an emission wavelength of 516 nm. For the antagonist assay, 50 µL of a 4-fold concentrated dilution series of scopolamine hydrochloride (positive control) and compounds was added. Subsequently an 80 µM stock solution of carbachol was added to all wells, and the relative fluorescence was measured with an excitation wavelength of 494 nm and an emission wavelength of 516 nm. Stock solutions of the compounds were in DMSO with a final concentration not exceeding 1% of DMSO.

Cells were seeded at a density of 2 × 10⁴ per well in a 96-well plate and allowed to attach overnight. The medium was replaced with fresh medium supplemented with desired concentrations of 5 for 6 h (MCF-7) and 12 h (Mia PaCa-2, PANC-1). In some experiments, cells were pretreated with 2-APB (30 μM) or BAPTA (10 μM). The Ca²⁺ level was evaluated using a Fluo-4 Direct Assay Kit (Invitrogen) according to the manufacturer’s instructions.