Intracellular calcium assay

CCK-stimulated biological responses were determined by measuring intracellular calcium responses. In brief, cells were seeded at a density of approximately 20,000 cells/well on a poly-lysine coated black clear-bottom 96-well plates. Cells were washed with calcium assay buffer (25 mM HEPES (pH 7.4), 104 mM NaCl, 5 mM KCl, 1.5 mM CaCl₂, 1.0 mM KH₂PO₄, 1.2 mM MgSO₄, 1.2 mM MgCl₂, 0.2% bovine serum albumin, 2.5 mM probenecid) and assays were initiated after cholesterol modification as described above and after loading with 0.75 μM Fluo-8-AM for 45 min at 37°C in the dark. Assays were performed by robotic addition of increasing concentrations of agonist ligand (0 to 100 μM) using a FlexStation 3.0 plate reader equipped with Softmax Pro 5.4 software (Molecular Devices, Sunnyvale, California). Intracellular calcium responses were determined by measuring the fluorescence emission intensity at 525 nm after excitation at 485 nm, with data collected every 4 s for 120 s. All assays were performed in duplicate and repeated in a minimum of 3 independent experiments. The peak calcium responses were analyzed and plotted as percentages of the maximal response to 0.1 mM ATP using nonlinear regression analysis in the Prism 9.2.