ELISA Quantification of Astrocyte Cytokines

ELISA was performed as already described [99 (link)]. Briefly, (after titration in increasing dilutions) the astrocyte culture supernatants were diluted in a 1:1 ratio by ELISA coating buffer (15 mM Na₂CO₃, 35 mM NaHCO₃, pH 9.6) and were used for the coating of 96 well polystyrene ELISA plates (Maxisorp, NUNC Intermed, Copenhagen, Denmark). Free binding capacity of the wells was blocked by 1% gelatin (Reanal, Budapest, Hungary) in PBS. After washing with PBS, anti-IL-6 [98 (link)] and anti-TNFα [100 (link)] primary antibodies were added (2 h, 37 °C), followed by goat-anti-rabbit-HRP secondary antibodies (1:2000, DakoCytomation, Glostrup, Denmark). Colour reaction was developed by o-phenylene-diamine, and the optical density was measured at λ = 492 nm by ELISA plate reader (Titertek, Uniscan, Flow Laboratories, Helsinki, Finland).
Optical density data were averaged, and the standard error of mean values was calculated for each treatment. Bar charts showed relative changes in the expression level of cytokines in percentages, and the control level was set to 100%. Graphs show representative data out of three independent experiments. Statistically significant differences were calculated by one-way ANOVA followed by Student-Newman-Keuls pairwise comparison. The difference between groups was considered statistically different if p < 0.05.