Cytosolic Calcium Signaling Assay

Cytosolic Ca²⁺ signal was determined at RT in cells loaded with 4.5 μM FURA-2·AM (Molecular probes Life technologies) (30 min) as previously described [50 (link)]. Cytosolic Ca²⁺ increases are represented as the normalized ratio of emitted fluorescence (510 nm) after excitation at 340 and 380 nm, relative to the ratio measured prior to cell stimulation (FURA-2 ratio 340/380). Cells were bathed in an isotonic solution (ISO) containing (in mM): 140 NaCl, 5 KCl, 1.2 CaCl₂, 0.5 MgCl₂, 5 glucose, 10 HEPES (300 mosmol/L, pH 7.4 with Tris). The following reactives were used during calcium experiments: 10 μM threo ifenprodil hemitartrate (If; a selective inhibitor of NMDA receptor having a GluN2B subunit; Tocris), 100 μM NMDA plus 100 μM Gly, 50 μM-(-) bicuculine methiodide (BIC; an antagonist of GABA A receptor; Tocris) plus 2.5 mM 4-aminopyridine (4-AP; a selective blocker of potassium channel Sigma).