Poly(I:C) promotes TNFa/TNFR1-dependent oligodendrocyte death in mixed glial cultures

Steelman, Andrew J; Li, Jianrong

Oligodendrocytes, microglia, astrocytes and mixed glial cultures were isolated from the forebrains of 1 to 2-dold Sprague Dawley rats or mice using a differential detachment procedure [28] . Briefly, after brain dissection and removal of the meninges the forebrains were digested with HBSS containing 0.01% trypsin and 10 μg/ ml DNase for 5 min at 37°C. The cells were washed twice with Dulbecco's Modified Eagle Media (DMEM) containing 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin. Following filtration through a sterile 70 μm filter to remove excess debris, the cells were counted and plated onto poly-d-lysine coated 75 cm 2 flasks or directly into 24-well plates for experiments using mixed glia. For mixed glial culture experiments, the medium was changed every other day and cells were treated after 7-8 days in vitro (DIV) in serum-free Basal Defined Medium (BDM: DMEM containing 0.1% bovine serum albumin, 50 μg/ml human apo-transferrin, 50 μg/ ml insulin, 30 nM sodium selenite, 10 nM D-biotin, and 10 nM hydrocortisone). For experiments involving mono and co-cultures, microglia were isolated by shaking the mixed glia-containing flasks for 1 h at 200 rpm. After removing microglia, the flasks were shaken overnight to separate preOLs from the astrocyte layer. Single cell suspensions from rat spleens [29] were used as a positive control for TLR3 western blot analysis. Briefly, the external capsule of the spleen was broken and cells passed through sterile 70 μm nylon mesh twice in DMEM containing 10% FBS. The cells were pelleted by centrifugation at 1500 × g for 5 min and the red blood cells were lysed with ammonium chloride (0.01 M) in PBS for 3 min at 24°C. The splenocytes were then washed in PBS.