Crl 2540

C8B4 murine microglial (MG) cell lines expressing the classical MG markers MAC1, F4/80, 2-4G2, Iba1 and TREM2 (but not GFAP) were cultured according to the manufacturer’s protocols (ATCC CRL-2540; Manassas VA) and previously published peer-reviewed work from our lab [12 (link),19 (link),21 (link),28 (link),29 (link)]. MG cell cultures were used at 3 days or 1 wk, and contained approximately 95% MG cells and 5% astroglial/oligodendroglial cells. They were prepared and analyzed according to established methods by the ATCC or our own laboratory [19 (link),26 (link),27 (link),29 (link)].

Murine C8-B4 microglia cells (ATCC CRL-2540; monocytes from cerebellum of C57BL/6 mice (Mus musculus) and C8-D1A astrocytes (ATCC CRL-2541; astrocyte type I clone from C57BL/6 mouse cerebellum) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and grown in DMEM-supplemented with 10% FBS and 1% penicillin/streptomycin. The microglia and astrocytes were cultured at 37 °C under 5% CO₂ in a humidified incubator (NuAire, Plymouth, MN, USA) and sub-cultivated by trypsination once/twice per week in a 1:5/1:10 split ratio, respectively. They were allowed to grow as monolayers in 75 cm² cell culture flasks with filter screwcaps (Techno Plastic Products, Trasadingen, Switzerland) until 80–100% confluence was reached. The microglia and astrocytes were plated at 90,000/cm² and 45,000/cm², respectively, 24 h prior to the test, which resulted in approximately 90% confluence at the day of exposure as observed by light microscopy (Leica DMIL, Solms, Germany). The cell culture medium was refreshed before performing the exposure test.

C8-B4 cells, a mouse microglial cell line, were purchased from the American Type Culture Collection (Rockville, MD, USA; ATCC^®, CRL-2540™). Opioid receptor agonists (DAMGO, DADLE, and U-50488), and lipopolysaccharide (LPS; Escherichia coli 055:B5), were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cell culture media, Hanks’ balanced salt solution (HBBS), fetal bovine serum (FBS), and disposable plasticware were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Protran (BA83 and BA85) nitrocellulose membranes were purchased from Schleicher & Schuell BioScience (Dassel, Germany). All other chemicals were from Merck KGaA (Darmstadt, Germany) and were of the highest available purity.