Spinning disc ix81 microscope

Hippocampal neurons cultured in round 35 mm coverslips at a density of 160,000 cells/coverslip were transfected with EGFP at 11 DIV. Then, at 14 DIV the neurons were placed in the imaging chamber in an isotonic solution (in mM: 1.2 CaCl₂, 140 NaCl, 2.5 KCl, 0.5 MgCl₂, 5 glucose, and 10 HEPES (305 mOsm/L, pH 7.4 with Tris)). The EGFP-positive neurons were imaged with an Olympus Spinning Disc IX81 microscope every 5 min for 45 min after the treatment with 1 μM Mas-7. The images were processed and analyzed using ImageJ software.

Intracellular Ca²⁺ measurements were carried out in DIV10 hippocampal neurons loaded with the ratiometric probe Fura-2 AM at 4.5 µM for 30 min. Before the assay, neurons were treated for 1 h with control or Wnt5a medium or with Wnt5a plus 1 µM HRI-i (JANSEN), 10 µM 7NI (SIGMA) or 250 nM sFRP2 (R&D systems), with a pretreatment of 30 min for all of these inhibitors. The experiments were performed with an isotonic solution containing (in mM): 2.5 CaCl₂, 132 NaCl, 4 KCl, 10 NaHCO₃, 6 glucose, and 10 HEPES (305 mOsm/liter, pH7.4). Cytosolic Ca²⁺ levels were continuously recorded (each 5 s) for 1 min before (basal levels) and 6 min after application of 100 µM NMDA + 100 µM glycine. Variations in cytosolic Ca²⁺ are presented as the normalized ratio of the emitted fluorescence at 510 nm after excitation at 340 and 380 nm relative to the ratio measured prior to stimulation (first minute before application of the stimuli), and the area under the curve (AUC) after the addition of NMDA and glycine was integrated. Live Ca²⁺ imaging was performed with an Olympus spinning disc IX 81 microscope.