To measure cytosolic or mitochondrial Ca2+ concentration, cells were cultured in white 96-well plates (Corning) and reverse-transduced with adenovirus containing either the mutated mitochondrial matrix-targeted (mtAEQmut) (Montero et al, 2000 (link)) or wild-type cytosolic aequorin (CytAEQ) (Brini et al, 1995 (link)) probes and incubated overnight at 37°C and 5% CO2. Cells were washed three times in BSS + Ca2+ (120 mM NaCl, 5.4 mM KCl, 0,8 mM MgCl2, 6 mM NaHCO3, 5.6 mM D-glucose, 2 mM CaCl2, and 25 mM HEPES [pH 7.3]) and incubated with 5 μM coelenterazine (Sigma-Aldrich) in BSS + Ca2+ for 90 min at 37°C and 5% CO2. Post-incubation, cells were washed once in BSS + Ca2+ and luminescence was measured by spectrophotometry (ClarioSTAR, BMG LabTek). Luminescence was measured every 2 s for 2 min. Basal luminescence was measured for 10 s followed by 100 μM histamine stimulation. At 1 min, cells were digitonized and saturated with Ca2+ by injection of 100 μM digitonin and 10 mM CaCl2, to discharge all luminous potential. Aequorin luminescence was calibrated into Ca2+ concentration using Equation (1). For mtAEQmut: n = 1.43, KTR = 22,008 and KR = 22,770,000. For CytAEQ: n = 2.99, KTR = 120 and KR = 7,230,000. Statistical significance was determined from four independent experiments (N = 4) by repeated measures one-way ANOVA and Tukey’s post hoc test for differences. Ca2+(M)=(LLMax×λ)1n+((LLMax×λ)1n×KTR)1KR((LLMax×λ)1n×KR) Equation (1). Relationship between Ca2+ concentration and AEQ luminescence. L = Light intensity, LMax = Sum of all light intensities, KR = Constant for Ca2+-bound state, KTR = Constant for Ca2+-unbound state, λ = Rate constant for AEQ consumption at Ca2+saturation. n = Number of Ca2+ binding sites (Bonora et al, 2013 (link)).
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