The firmly attached microglia were then trypsinized and seeded on poly-l-lysine-coated coverslips sitting in 12-well plates. To prepare OPCs, the flasks were shaken again at 200 rpm overnight. The supernatant was transferred to a noncoated Petri dish and incubated at 37°C for 1 h. The Petri dish was gently swirled to release loosely attached cells and the supernatant was transferred into poly-l-lysine-coated flasks. OPCs were maintained in a chemically defined medium (CDM), which contains DMEM/F12, 0.1% BSA, 100 μmol/L Inhibitors,research,lifescience,medical putrescine, 20 nmol/L progesterone, 10 nmol/L sodium selleckchem selenium, 20 nmol/L biotin, 5 μg/mL cysteine, 5 nmol/L hydrocortisone, 5

μmol/L insulin, 50 μmol/L transferrin, 2 nmol/L l-glutamine, and penicillin/streptomycin), supplemented with PDGF-AA and bFGF (final concentration 10 ng/mL, each). To enrich astrocyte population, the remaining cells in the flasks were shaken again for 6 h at 220 rpm, and the supernatant was discarded. Cells remained Inhibitors,research,lifescience,medical in the flasks were mostly astrocytes, which were further subcultured with a 1:6 split ratio, and fed with DMEM/20% FBS. When astrocytes reached 80% confluence, they were trypsinized and seeded onto coverslips sitting in 12-well plates at a density of 1 × 105 per well. The Inhibitors,research,lifescience,medical purity of glial cultures was assessed by immunocytochemistry using glial specific markers

(i.e., NG2 and O4 for OPCs, GFAP for astrocytes, and CD-11b for microglia). Typically, the purity of OPC, astrocyte, and microglia cultures was 95.4%, 97.7%, and 94.2%, respectively (estimated from three separate primary cultures). Neuron-OL myelination coculture The coculture was prepared from the cortex of embryonic day 16 rat fetuses, as described elsewhere (Pang et al. 2012).

Inhibitors,research,lifescience,medical Briefly, the cortices were collected in cold Hank’s balanced salt solution (HBSS) and chopped into small pieces using a surgical Inhibitors,research,lifescience,medical blade. Tissue was digested in trypsin/ethylenediaminetetraacetic acid (EDTA) solution for 10 min at 37°C. The reaction was stopped by addition of trypsin inhibitor-DNase-I solution, and tissue was dissociated into single cell suspension by passing through 1 mL fine pipette tip for five times. Cells were then seeded on poly-l-lysine-coated coverslips at a density of 0.4 × 105 per cm2. Cultures were maintained isothipendyl in N2-NBM (1:1) with nerve growth factor (NGF) (50 ng/mL) and neurotrophin-3 (NT-3) (10 ng/mL) for the first 7 days of culture, and then in insulin-free N2-NBM (4:1) afterwards. We have previously shown that extensive myelination could be detected in the cocultures by 4 weeks (Pang et al. 2012). Preparation of the conditioned medium and treatment In order to compare the effects of the two types of glia-conditioned medium on OL phenotypes, the density of astrocytes and microglia as well as the volume of the medium were kept identical.