Calcium Imaging of Cell Responses

RG17 and XG69 cells were plated on glass cover slips and incubated in the calcium indicator Fluo-4, acetoxymethyl ester in complete media for 30 minutes. The media were then aspirated, and cells were immersed in physiological buffer (125 mmol/L NaCl, 5.9 mmol/L KCl, 2.56 mmol/L CaCl₂, 1 mmol/L MgCl₂, 25 mmol/L HEPES, and 0.1% bovine serum albumin, pH 7.4) for imaging on a Leica TCS SP5 upright confocal microscope (Leica Microsystems). Adenosine triphosphate (ATP) disodium (50 µM; Selleck Chemicals, Houston, TX, Cat# S1985), KN-62 (1 µM; Selleck Chemicals, Cat# S7422), 2′-3′-O-(4-benzoylbenzoyl) adenosine 5′-triphosphate (50 µM; BzATP) triethylammonium salt (Tocris, Minneapolis, MN, Cat# 3312), and glutamate (100–1000 µM) were used. Cytosolic calcium levels were quantified as described by Makhmutova et al.²⁰ (link) Changes in fluorescence intensity were expressed as percentage changes over baseline (ΔF/F). Baseline was defined as the mean of the intensity values during the nonstimulatory period. Data are displayed as heat maps to include all recorded cells from two to three independent cultures. To measure the overall integrated calcium response, the area under the curve for each response was calculated with GraphPad Prism 6 v9.0.2 (San Diego, CA).