Figure 2.

Purity validation of fetal sheep brain primary microglia in cultures and LPS second hit. (A) Photomicrographs (ICC) confirming cell purity. Iba1+ staining in microglia vs. undetectable GFAP signal in microglia indicating no contamination with astrocytes in the culture. Microglia were cultured in eight-well chamber slide with DMEM +5% FCS for 4–7 days. Microglia were collected from the floating fraction and stained for Iba1 and GFAP. Scale bar = 50 μm. Magnification 40× for both images. (B) Purity of fetal sheep brain primary microglia cultures was verified by flowcytometry. After several days in culture, fetal sheep microglia were scraped from the wells using a cell scraper and blocked for 30 min using normal mouse IgG and 10% human serum. Cells were then stained using a FITC-conjugated monoclonal bovine anti-CD11b (1:40, Bio-Rad) on ice for 20 min. Cells were washed in FACS buffer and analyzed using a BD FACSCalibur and FlowJo software. (C) Microglia from in vivo LPS exposed brain appear more aggregated in vitro than microglia derived from in vivo controls. Microglia were cultured in 24-well plates with DMEM +5% FCS for 4–7 days, when images were taken. Cells were extracted from a twin control fetal brain and an in vivo LPS exposed fetal brain (Magnification 20× for both images). (D) Effect of “second-hit” in vitro LPS treatment on microglial phenotype. BOTTOM: IL-1β concentration in conditioned media of microglia derived from fetal sheep brain that were exposed to LPS vs. saline in vivo (^***P < 0.0001). Cultures from in vivo LPS-exposed (SH), n = 4, cultures from in vivo Control (Naïve), n = 10. Cell culture media supernatant was obtained by centrifugation upon cell culture termination. A sheep specific IL-1β ELISA was performed to measure cytokine levels in cell culture media. In vitro, at baseline, microglia secreted more IL-1β in the in vivo LPS group (SH_C) than in naïve Control (N_C). LPS re-exposure (SH_L) further increased IL-1β vs. naïve LPS (N_L) by ~4.6-fold.