Cell culture and LPS stimulation

Mouse BV2 microglia phenotype cells and mouse J774 macrophage cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) with high glucose (4500 mg/l), l-glutamine, sodium pyruvate, and sodium bicarbonate (D6429 Sigma) that was supplemented with 10% fetal bovine serum (12003C Sigma; Australian Origin), and 1% antibiotic-antimycotic (10,000 IU/ml penicillin, 10,000 μg/ml streptomycin, and 25 μg/ml of Fungizone® Antimycotic; 15240062 Life Technologies). The murine BV2 microglia cells were a generous gift from Prof. Gilles Guillemin (Macquarie University, Sydney, Australia), and J774 cells were a generous gift from Dr. Nisha Schwarz (SAHMRI, Adelaide, Australia). Cells were maintained in a humidified incubator with 95% air and a 5% CO₂ atmosphere at 37 °C. All cells were sub-cultured for ISH experiments onto sterile, RNAse-free coverslips (22 mm × 22 mm) for 24–48 h inside six-well plates. The cells were incubated with LPS (0.5 μg/ml; tlrl-3pelps LPS-EB Ultrapure; InvivoGen) for the specified times under normal culture conditions.