Dg 4 wavelength switcher

These experiments were conducted with the Ca²⁺ probe Fluo4 as previously described [13 (link),16 (link),17 (link)]. Briefly, cells were loaded with 5 µM Fluo4/AM for 25 min, washed, and kept for another 5–10 min in a standard recording saline solution containing (in mM) 150 NaCl, 5 KCl, 1 MgCl₂, 2 CaCl₂, 5.5 glucose, and 10 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.4). All these procedures were performed at room temperature and in the dark. Coverslips were then mounted on the stage of an inverted Axio Observer A1 microscope with a Fluar 40× oil immersion objective lens (1.3 NA) (Carl Zeiss, Marly le Roi, France) and a charge-coupled device camera (CoolSnap HQ2, Princeton Instruments, Roper Scientific, Evry, France). The experimental setup, equipped with a DG-4 wavelength switcher (Princeton Instruments, Roper Scientific, France), was driven by MetaFluor (Universal Imaging, Roper Scientific, Evry, France). The cytosolic Ca²⁺ signals were acquired from cells bodies at a sampling rate of 0.2 Hz.

Primary cultures of cortical neurons were prepared from embryonic (E13) C57BL6/J mice as previously described (Bouron et al., 2005 (link)) and approved by the animal care committee of the CEA's Life Sciences Division (CEtEA). Experiments were conducted in accordance with French legislation and the European Community Council Directive of 24 November 1986 (86/609/EEC). Calcium imaging experiments were performed at room temperature as previously described (Gibon et al., 2013 (link); Chauvet et al., 2015 (link), 2016 (link)). Briefly, cortical neurons were incubated in a saline solution containing (mM) 150 NaCl, 5 KCl, 1 MgCl₂, 2 CaCl₂, 5.5 glucose, 10 HEPES (pH 7.4) supplemented with 5 μM Fluo4/AM. After 20 min of incubation, neurons were rinsed twice and incubated for 10 min in a Fluo-4/AM-free saline. The imaging system consisted of an inverted Axio Observer A1 microscope equipped with a Fluar 40 × oil immersion objective lens (1.3 NA) (Carl Zeiss, France) and a CCD CoolSnap HQ2 camera (Princeton Instruments, Roper Scientific, France). A DG-4 wavelength switcher (Princeton Instruments, Roper Scientific, France) was used with λ_EX = 470 nm and λ_EM = 525 nm. The setup was driven by MetaFluor (Universal Imaging, Roper Scientific, France).