Glast 1 apc

Microglia and astrocytes were Percoll enriched as described above. Microglia and astrocyte fractions were pooled after isolation and were labeled with rat anti-mouse CD11b-FITC, CD45-PerCP-Cy5.5 (eBioscience) and GLAST-1-APC (Miltenyi Biotec) antibodies. Cells were sorted using a Becton-Dickinson FACSAria III cell sorter at the OSU Comprehensive Cancer core facility. Microglia were identified by GLAST-1⁻/CD11b⁺/CD45^low expression and astrocytes were identified by GLAST-1⁺/CD11b⁻/CD45⁻ expression. CD45^high expressing peripherally derived macrophages were excluded when collecting microglia. Microglia and astrocytes (100,000–150,000 cells) were sorted into separate collection tubes. Cells were pelleted and lysed immediately in RNA lysis buffer.

In a separate cohort of mice, astrocytes were Percoll enriched as described above and were labeled with rat anti-mouse CD11b-FITC, IL-10R1-PE (eBioscience), and GLAST-1-APC (Miltenyi Biotec) antibodies (Norden, et al., 2014 (link)). Expression was determined using a Becton-Dickinson FACSCaliber four color Cytometer. Astrocytes were identified by GLAST-1⁺/CD11b⁻ expression. In this experiment, CD11b was used to exclude both microglia and any peripherally derived myeloid cells from the astrocyte analysis. For each antibody, gating was determined based on appropriate negative isotype stained controls. Flow data were analyzed using FlowJo software (Tree Star, CA).