Vented caps

HeLa, Ramos, and CCL-119 cells were obtained from the American Type Culture Collection (ATCC). HeLa cells were cultured in MEM-α (Thermo) supplemented with 10% (v/v) fetal bovine serum (FBS, Thermo). CCL-119 and Ramos cells were cultured in RPMI 1640 (Thermo) supplemented with 10% (v/v) FBS. MDA-MB-231/Luc human breast cancer cell line purchased from Cell Biolabs Inc. was grown in DMEM (High Glucose) supplemented with 10% FBS, 0.1 mM MEM Non-Essential Amino Acids (NEAA) and 2 mM l-glutamine. All cells were grown in T75 cm² flasks with vented caps (Sarstedt) in a humidified incubator at 37 °C with 5% CO₂. STR (short tandem repeat) profiling and mycoplasma test were done for all cell lines in culture. No contamination was found in any of the cell lines used for experiments.

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.