Ca2+ imaging

Ca²⁺ imaging in fura-2/AM loaded HEK293T cells was performed as previously described [22]. Briefly, dye-loaded cells were washed and imaged using an inverted microscope (Leica DMI4000B, Leica Microsystems) equipped for ratiometric live-cell imaging with a Visichrome polychromator system (Visitron Systems) for multiwavelength excitation, a 12-bit 1392 x 1040 CCD camera (CoolSnap EZ, Photometrics), and LAS MMAF software (Leica Microsystems). Cells were illuminated sequentially at 340 and 380 nm (1 Hz cycles) and average intensities within user-selected regions of interest were used to calculate f₃₄₀/f₃₈₀ intensity ratios.

Ca²⁺ concentrations [Ca²⁺] were calculated according to [40] using the equation [Ca²⁺] = K_d * (F₀/F_s) * [(R − R_min)/(R_max − R)]. K_d is the fura-2 dissociation constant (224 nM), F₀ and F_s are coefficients for free and Ca²⁺-bound fura-2, respectively (measured at 380 nm), and R_min and R_max were determined by applying 5 μM of ionomycin in [Ca²⁺]_zero (10 mM of EGTA) and saturating Ca²⁺ (10 mM). Values for R_min, R_max, and F₀/F_s were 0.56, 0.85, and 1.88, respectively.