CD73 Monoclonal Antibody (AD2), APC, eBioscience™

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	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.

Protocol tips
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.

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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