Fura-2/AM Fluorescence for Cytosolic Ca2+ Measurement

To determine cytosolic Ca²⁺ concentration ([Ca²⁺]_i), Fura-2/AM fluorescence was utilized^{60 (link)–65 (link)}. Cells were preincubated for 15–30 minutes with Fura-2/AM (2 µM, Invitrogen, Goettingen, Germany) at 37 °C and excited alternatively at 340 nm and 380 nm in an inverted phase-contrast microscope (Axiovert 100, Zeiss, Oberkochen, Germany) through an objective (Fluor 40×/1.30 oil). At 505 nm, the emitted fluorescence intensity was recorded. Data (6/minute) were acquired using a computer software Metafluor (Universal Imaging, Downingtown, USA). To estimate cytosolic Ca²⁺ activity, a ratiometer (340 nm/380 nm) based analysis was employed. SOCE was determined following extracellular Ca²⁺ removal causing store depletion and subsequent Ca²⁺ re-addition in constant presence of SERCA inhibitor thapsigargin (1 µM, Invitrogen,Goettingen, Germany). For quantification of Ca²⁺ entry, the slope (delta ratio/s) and peak (delta ratio) were determined following re-addition of Ca²⁺. Experiments were performed with Ringer’s solution containing (in mM): 125 NaCl, 5 KCl, 1 CaCl₂, 32 HEPES, 2 Na₂HPO₄, 1.2 MgSO₄, 5 glucose, pH 7.4. Ca²⁺-free conditions were achieved by using Ca²⁺-free Ringer solution containing (in mM): 125 NaCl, 5 KCl, 1.2 MgSO₄, 2 Na₂HPO₄, 32 HEPES, 0.5 EGTA, 5 glucose, pH 7.4.