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

Steelman, Andrew J; Li, Jianrong

Cell viability following TLR agonist treatment was assessed using both immunocytochemical and biochemical techniques. Lactate dehydrogenase (LDH) activity was assessed from culture supernatants according to the manufacturer's instructions (Roche Applied Science; Indianapolis, IN). The effects of treatment on preOL viability in mixed glial cultures were determined 48 h after treatment with Pam3CSK4 (1.0 μg/ml; InvivoGen, San Diego, CA), poly(I:C) (50 μg/ml and 5 μg/ml), LPS (1.0 and 0.1 μg/ml) or R848 (5.0 μg/ml; InvivoGen, San Diego, CA) by counting the number of normal appearing nucleated O4 + cells per 200× magnification from 5 random fields per culture well. O4cells most likely representing microglia and astrocytes were excluded from the analysis. Pictures were taken using an Olympus DP70 digital camera mounted on an Olympus IX71 microscope. All treatments were conducted in at least triplicate, corresponding to between 750-1000 cells counted per treatment condition per experiment. Experiments were replicated at least three times. For each experiment the average number of O4 + cells in the control group was used to obtain percent reduction, and the percent reduction averaged across experiments. The effects of various treatments in pure preOL cultures were assessed by Alamar blue assay as described previously [24] .