Tcrγδ fitc

After sacrificing the experimental mice at 72 hours after ICH, brain peri-hematoma tissue was harvested and suspended in RPMI-1640 medium. The tissue was then digested in a shaker at 37°C, rotating at 950 × g, for 5 minutes with the addition of type IV collagenase (1 mg/mL) and DNase I (50 mg/mL). Cell supernatants were then removed, resuspended, isolated by centrifugation, and washed with RPMI-1640 prior to flow cytometry. The final cell amount was equivalent in both groups, with a concentration of 2 × 10⁶/mL. Cells from brain peri-hematoma tissue were incubated with fluorescence-labeled antibodies against the surface markers IL-17–APC-Cy7 (Cat. No. 560821; BD Biosciences, San Jose, CA, USA) and TCR γδ–FITC (Cat. No. 559501; BD Biosciences). Incubations were carried out according to the manufacturer’s protocols, at 4°C for 40 minutes in the dark.
In addition, TCR γδ cells were isolated from circulating peripheral blood mononuclear cells of C57BL/6 mice. After cell suspension of peripheral blood mononuclear cells, TCR γδ–FITC antibody was added to the suspension and incubated in the dark for 40 minutes. The infiltration of the fluorescence-labeled suspension was then tested. Fluorescence-activated cell sorting analysis was performed using a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA), and data were analyzed using FlowJo software 7.6.1 (FlowJo LLC, Ashland, OR, USA).