4.7. Ca2+ Measurements

Cells were seeded on glass cover slips and loaded with 2 µM Fura-2/AM and 0.02% pluronic F-127 (Invitrogen, Darmstadt, Germany) in Ringer solution (mmol/L: NaCl 145; KH₂PO₄ 0,4; K₂HPO₄ 1,6; Glucose 5; MgCl₂ 1; Ca²⁺-Gluconate 1,3) for 1 h at room temperature. Fluorescence was detected in cells perfused with Ringer’s solution at 37 °C using an inverted microscope (Axiovert S100, Zeiss, Oberkochen, Germany) and a high speed polychromator system (VisiChrome, Puchheim, Germany). Fura-2 was excited at 340/380 nm, and emission was recorded between 470 and 550 nm using a CoolSnap camera (CoolSnap HQ, Visitron). [Ca²⁺]_i was calculated from the 340/380 nm fluorescence ratio after background subtraction. The formula used to calculate intracellular calcium ([Ca²⁺]_i) was [Ca²⁺]_i =Kd × (R − R_min)/(R_max − R) × (S_f2/S_b2), where R is the observed fluorescence ratio. The values R_max and R_min (maximum and minimum ratios) and the constant S_f2/S_b2 (fluorescence of free and Ca²⁺-bound Fura-2 at 380 nm) were calculated using 1 µmol/L ionomycin (Calbiochem, La Jolla, CA, USA), 5 µmol/L nigericin (Sigma-Aldrich, St. Louis, MO, USA), 10 µmol/L monensin (Sigma-Aldrich, St. Louis, MO, USA), and 5 mmol/L EGTA to equilibrate intracellular and extracellular Ca²⁺ in intact Fura-2-loaded cells. The dissociation constant for the Fura-2•Ca²⁺ complex was taken as 224 nmol/L. Control of experiment, imaging acquisition, and data analysis were done with the software package Meta-Fluor (Universal Imaging, New York, NY, USA).