Control or A1 reactive astrocytes (see above) were grown for 7 d in serum-free media supplemented with 5 ng/ml HBEGF18. Cells were then treated with Il-1α, TNFα, and C1q or an equivalent volume of 1× dPBS and cells left for an additional 24 h. At this time, conditioned media was collected with cOmplete™, Mini, EDTA-free protease inhibitor cocktail (Sigma/Roche, 04693159001) and concentrated at 30kDa with Amicon Ultra-15 Centrifugal Filter Units (Millipore, UFC903024) until approximately 30–50× concentrated. A Bradford assay was performed to determine total protein concentration, and 1–50 μg/ml total protein was added to purified cell cultures of neurons, oligodendrocytes, OPCs, endothelial cells, astrocytes, pericytes or microglia (plated at 1,000 cells/well in poly-d-lysine-coated (PDL) 96-well plates, grown for 5 days in serum-free base media) and viability assed using the LIVE/DEAD® Kits for mammalian cells (Thermo Fisher Scientific, L3224). Additional experiments were done on RGCs and oligodendrocytes using heat inactivated A1 ACM (20-minute treatment at 60 °C) or protease treatment of A1 ACM (0.01 U/ml plasmin from human plasma, Sigma – P1867), 2 h at room temperature. Protease treatment was halted
