Publication protocol
After mouse dissections, brain structures were removed, the meninges were carefully stripped off and a single cell suspension was prepared (Pösel et al., 2016). The tissues were washed in phosphate buffered saline (PBS) containing 0.6% glucose, and the dissected tissue was minced and sieved through a 100-μm cell strainer. The strained cells were centrifuged at 286 × g for 5 min at 4°C, and the supernatant was carefully discarded. The pellet was resuspended in 1 ml of digestion buffer (Liberase with a low thermolysin concentration (up to 2 U/ml) in Hank’s balanced salt solution), and the suspension was incubated under slow continuous rotation at 37°C for 1 h. The cell suspension was sieved through a 70-μm cell strainer and rinsed thoroughly with 3 ml of HBSS containing DNase. The cell suspension was centrifuged at 286 × g for 5 min, and the supernatant was discarded. The cell pellet was resuspended in 5 ml of 25% density gradient medium of iodixanol solution (OptiPrep, Sigma-Aldrich, United States) and centrifuged at 521 × g for 20 min at 18°C. The myelin layer and the supernatant were aspirated, and the pellet was washed with 10 ml of HBSS. Cells were resuspended in flow cytometry staining buffer, incubated with anti-mouse CD16/CD32 antibodies to block FcR, and then subjected to staining with different combinations of cell surface markers and CD73 and analyzed by flow cytometry. Anti-mouse GLAST, CD11b, and O4 antibodies were used to label astrocytes, oligodendrocytes and microglial cells, respectively, and neurons were screened out as the GLAST-CD11b-O4-CD45-CD24+ population.
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