Aequorin-based Ca2+ Imaging in Cells

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 CaCl_2, to discharge all luminous potential. Aequorin luminescence was calibrated into Ca²⁺ concentration using Equation (1). For mtAEQmut: n = 1.43, K_TR = 22,008 and K_R = 22,770,000. For CytAEQ: n = 2.99, K_TR = 120 and K_R = 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. ${Ca}^{2+}\left(M\right)=\frac{{\left(\frac{L}{L_{\mathit{Max}}}\times \lambda \right)}^{\frac{1}{n}}+\left({\left(\frac{L}{L_{\mathit{Max}}}\times \lambda \right)}^{\frac{1}{n}}\times K_{TR}\right)-1}{K_R-\left({\left(\frac{L}{L_{\mathit{Max}}}\times \lambda \right)}^{\frac{1}{n}}\times K_R\right)}$ Equation (1). Relationship between Ca²⁺ concentration and AEQ luminescence. L = Light intensity, L_Max = Sum of all light intensities, K_R = Constant for Ca²⁺-bound state, K_TR = 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)).