Hank s buffered salt solution

Primary culture of rat ENS were generated using pregnant Sprague–Dawley rats (Janvier Laboratories SA, Le Genest-St-Isle, France) as previously described [11 (link)]. All housing and experimental procedures were carried out in compliance with the local ethical review panel of INSERM (agreement E. 44,011; INSERM, Nantes, France). Pregnant rats were killed by an overdose of CO₂ followed by severing the carotid arteries. The small intestines of rat embryos were removed, diced in Hank’s Buffered Salt Solution (Sigma, Saint-Quentin Fallavier, France) and collected in 5 mL of Dulbecco’s modified Eagle’s medium (DMEM)-F12 (Gibco®, Life Technologies, Villebon sur Yvette, France) (1:1) for digestion at 37 °C for 15 min in 0.1% (v/v) trypsin (Sigma). The trypsin reaction was stopped by adding medium containing 10% fetal calf serum and then treatment with DNase I 0.01% (v/v) (Sigma) for 10 min at 37 °C. After triturating with a 10 mL pipette, cells were centrifuged at 750 rpm for 10 min. Cells were counted and then seeded at a density of 2.4 × 10⁵ cells/cm² on 24-well plates previously coated with a solution of 0.5% (v/v) gelatin in sterile phosphate buffered saline. After 24 h, the medium was replaced with a serum-free medium DMEM-F12 (1:1) containing 1% (v/v) of N-2 supplement (Life Technologies). Cultures were maintained for 14 days.

Primary mixed glial cultures were prepared from P1 to P3 C57bl6 (WT) and caspase-6 KO mice of both sexes, as previously described [32 (link)], with some alterations. Briefly, brains were isolated and the cerebellum removed and washed in cold Hanks buffered salt solution (Sigma-Aldrich, USA) supplemented with 100 UI/ml penicillin and 100 μg/ml streptomycin (Sigma-Aldrich, USA). Forebrains were gently dissociated by titration in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich, USA) supplemented with 20% heat-inactivated fetal bovine serum (FBS; Gibco; Life Technologies Sweden) and 1% antibiotics. The cell suspension was passed through a 70-μm filter (Becton-Dickinson) and plated in 75-cm² flasks with vented caps (Sarstedt, Germany) at a density of two brains per flask. After 7 days all the media was replaced with DMEM/10% FBS/antibiotics and cultured for a further 7 days. Microglia were then detached by shaking (250 rpm, for 3 h at 36°C) on a rotary shaker, and the microglia cell suspension was collected and centrifuged (250 g × 10 min). The media were then removed, the pellet was resuspended in DMEM/2% FBS/antibiotics and the number of cells were counted with an automated cell counter (Scepter; Millipore) and seeded into 12-well plates (approximately 2-2.5 × 10⁵ cells per well). Flasks and plates were incubated in a humidified oven at 37°C containing 5.1% CO₂ in air.