Attune nxt software version 2

Fresh cerebrovascular tissue was isolated from the brains of r-sham and r-mTBI mice at 6 months post-last injury and processed as previously described (Crouch and Doetsch, 2018 (link)). Briefly, the cerebrovascular pellet (obtained as described above) was resuspended in a collagenase/dispase solution (1 mg/ml) and incubated for 1 h at 37 °C with gentle agitation. Following enzymatic digestion, the tissue was pelleted by centrifugation at 6000 rpm for 3 min and resuspended in a DNAse solution (1 mg/ml, Worthington Biochemical) and subjected to further mechanical digestion via trituration with a pipette to achieve a single cell suspension. The tissue was centrifuged at 6000 rpm for 3 min, the supernatant discarded, and the resulting pellet was resuspended and stained with antibodies against the mural cell-specific N-aminopeptidase (CD13) using anti-CD13-FITC at 1:200 (BD Biosciences), and the brain endothelial cell marker (CD31) using anti-CD31-PE-CY7 at 1:500 (BD Biosciences). For live/dead cell discrimination, the viability dye propidium iodide (Sigma Aldrich) was added to the antibody cocktail. Cells were stained on ice for 30 min in the dark and resuspended in 1% BSA in HBSS. Data were acquired and analyzed using the Attune® NxT Acoustic Focusing Flow Cytometer and Attune® NxT software version 2.7 (Thermo Fisher Scientific, Waltham, MA, USA).