Activated primary astrocytes-microglia cultures from Eif2b5^R132H/R132H mice do not exhibit increased global translation and display impaired production of cytokines.

<p>Primary mixed glia cultures were isolated from Eif2b5^R132H/R132H (Mut) and wild-type (WT) mice. (A) Representative analysis of PE-labelled anti-CD11b staining followed by flow cytometry analysis. Both Mut and WT cultures contained 93–95% CD11b-negative (gray area) cells. (B) Immunoblot analysis of 3 independent experiments using anti-TLR4 and anti-β-actin antibodies. No statistically significant difference between WT and Mut cells was observed. (C) WT and Mut cells were treated with 2 µg/ml LPS for 48 h and metabolically labelled with [³⁵S]L-methionine and [³⁵S]L-cysteine for 30 min followed by determination of [³⁵S]-Met/Cys incorporation following trichloroacetic acid precipitation. The incorporation level (cpm/µg protein) in untreated WT cells was set at 100%. Bars represent the means ± SEM of 3 independent experiments, *p<0.02. (D, E) WT and Mut cultures were incubated with 2 µg/ml LPS for the indicated times followed by cell harvest and media collection. A representative immunoblot analysis of intracellular IL-6 and IL-1β protein levels is shown and the average (of 3 independent experiments) of IL-6/β-actin and IL-1β/β-actin in Mut relative to WT is provided in (D); protein concentrations of IL-6, TNF-α and MCP-1 in the media was measured by ELISA and a representative of 3 independent experiments is shown in (E).