Ca2+ imaging

Cells grown on 25-mm glass coverslips were washed and then loaded with 7 µM Fura-2 AM (acetoxymethyl ester, #F1221; Thermo Fisher Scientific) with 0.02% pluronic F-127 (#P3000MP; Thermo Fisher Scientific) for 45 min at 37°C. Cells were washed, mounted in a perfusion chamber, and visualized using a ×40 objective on a Nikon Diaphot microscope (Nikon, Melville, NY, USA) as described (30). Ratiometric measurements were performed by alternating the excitation wavelength from 340 to 380 nm and quantifying emission ≥512 nm with a charge-coupled device camera (All Photon Technologies International, Lawrenceville, NJ, USA). Cells were perfused with isotonic solution containing 105 mM NaCl, 5 mM KCl, 6 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 4 mM Na 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 5 mM NaHCO₃, 60 mM mannitol, 5 mM glucose, 1.3 mM CaCl₂, and 0.5 mM MgCl₂. The ImageJ (NIH; https://imagej.hin.gov/ij/) Pseudocolor Look-Up Table (31) was used for displaying images but not for quantification. Because calibration was frequently complicated by cellular manipulations, most of the data were expressed as the ratio of light excited at 340–380 nm (F_340/380), em >540 nm. However, levels of cytoplasmic calcium were calibrated in some trials as previously reported (32), using 5 µM ionomycin in isotonic (high Ca²⁺) or Ca²⁺-free + 5 mM EGTA, both at pH 8.0, based on the equation Ca²⁺ (nM) = K_d*Sf2/Sb2*(R − R_min)/(R_max − R), where K_d = 350 nM, Sf2 is the fluorescence at 380 nm in Ca²⁺-free and Sb2 in high Ca²⁺ solution, R_min is 340/380 nm in Ca²⁺-free, and R_max is 340/380 in high Ca²⁺. Additional experiments were performed on cells grown to confluence in 96-well black-bottom plates, loaded with Fura-2 AM as previously described, and fluorescence was measured in a Fluoroskan Ascent plate reader (Thermo Fisher Scientific) as described in Guha et al. (33). Autofluorescence was subtracted from wells when possible.