Figure 5

IL-17RA expression in astrocytes and microglia in vitro. (A-C) Phenotypic characterization of glial cells by immunofluorescence. Mixed glial cultures (A), and purified astrocytic (B) and microglial cultures (C) were established from neonatal C57BL/6 mice. Cultures were stained with anti-GFAP antibody (astrocytic marker; green) and anti-CD11b (microglial marker; red) and counterstained with nuclear stain DAPI (blue). Mixed glial cultures primarily consist of astrocytes (70–80%) and microglia (5–10%); whereas, purified astrocyte cultures consist of 80–90% GFAP-positive cells. Purified microglial cultures are 98–99% CD11b-positive. (D-F). Flow cytometry. Glial cells were immunostained for flow cytometric analysis. Mixed glial cultures (D) contain both GFAP- and CD11b-positive cells. Astrocyte cultures were free from microglia (< 0.5%) (E) and microglial cultures free of astrocytes (< 0.5%) (F). (G) IL-17RA expression in vitro. mRNA was extracted from glial cultures and IL-17RA expression was quantified by RT-PCR using a primer set from exon boundary 1–2. Data represent the mean ± SEM expression of total IL-17RA mRNA from isolated cultures from three different batches of donors. IL-17RA is expressed 4-fold higher in microglia compared to astrocytes (***p < 0.0001). Mixed glial culture confers more expression of IL-17RA mRNA in comparison to astrocyte cultures devoid of microglia (*p = 0.0329).