Fcs express 6

Tissue dissociation for flow cytometry was adapted from 83, 88 . Mice were deeply anesthetized and perfused with 20 mL of DPBS. Spinal cords and brains were isolated. Brains were sectioned into coronal slices and the cortex, hippocampus, cerebellum, and striatum were isolated. The number of animals differs between regions because in some animals only the spinal cord and cerebellum were analyzed. Separated tissue was minced and placed in 1 mL of accutase for 40 min at 4 °C. Tissue was resuspended in Hibernate A medium (Brain Bits, LLC) and transferred to a 2 mL Dounce homogenizer and tissue was disrupted until no large pieces remained. Cell suspension was filtered using a 100 μm cell strainer. Isolated cells were stained for flow cytometry (BD Aria) for the following markers: Hoechst, Live/Dead stain, FITC anti-mouse ACSA-2, APC TGFBRII, APC TNFR, APC IL17RA, PE IFNGR1, PE IL1R1, and PE IL10RA. An unstained control and single stain control for each fluorophore were used to identify the cell population of interest, set flow cytometer voltage, and calculate compensation. Flow cytometry data was analyzed using FCS Express 6 or FlowJo.