Dual excitation spectrofluorometer

Intracellular Ca²⁺ was measured using the Ca²⁺-sensitive dye Fura-2 and a dual-excitation spectrofluorometer (IonOptix LLC, Milton, MA, USA). Briefly, cells were loaded with 2.5 μM Fura-2 for 20 min at room temperature in Tyrode’s buffer containing (in mM): 144 NaCl, 1 MgCl₂, 10 HEPES, 5.6 glucose, 5 KCl, 1.2 NaH₂PO₄, 1.5 CaCl₂ (adjusted to a pH 7.4 with NaOH). Then the cells were washed with fresh regular Tyrode’s solution for at least 10 min. Cells were excited with a xenon lamp at 340 nm and 380 nm wavelengths. The emission fluorescence (510 ± 15 nm) was collected by a photomultiplier and subsequently integrated by the system’s interface. The Ca²⁺ signal was recorded under spontaneous cardiomyocyte contractile activity or electric field-paced (20 V) at different frequencies from 0.5 to 4 Hz. The calibration was performed in cardiomyocytes by superfusing a free Ca²⁺ and then a Ca²⁺ saturating (5 mmol/L) solutions, both containing 10 μmol/L ionomycin (Sigma, St. Louis, MO) until reaching a minimal (R_min) or a maximal (R_max) ratio value, respectively. (Ca²⁺)_i was calculated as previously in Dulce et al.^{[3 (link)]} using the following equation:
$${\left[C{a}^{2+}\right]}_i=K_d\times \frac{S_{f2}}{S_{b2}}\times \frac{\left(\text{R}-R_{\text{min}}\right)}{\left(R_{max}-\text{R}\right)}$$
K_d (dissociation constant) in adult myocytes was taken as 224 nmol/L. The scaling factors S_f2 and S_b2 were extracted from calibration^{[3 (link)]}.