γδtcr gl3

Flow cytometry analysis was performed according to the methods described previously [28 (link)]. Briefly, the cells isolated from the MLN (the MLN cells) were incubated with an Fcγ receptor-blocking mAb (CD16/32; 2.4G2, BD Biosciences) for 15 minutes at 4°C. For surface antigen detection, the cells were labeled with monoclonal antibodies against Gr-1 (RB6-8C5, BD Biosciences), F4/80 (BM8, BD Biosciences), αβTCR (H57-597, BD BioLegend), γδTCR (GL3, BioLegend), NK1.1 (PK136, BioLegend), CD4 (RM4-5, BD Biosciences), CD44 (IM7, BD Biosciences), CD25 (PC61, BioLegend), B220 (RA3-6B2, BioLegend), and CD19 (MB19-1, BioLegend) for 30 min at 4°C.
For intracellular cytokine staining, the cells isolated from the MLN (the MLN cells) were stimulated with ionomycin (1 mg/mL; Sigma-Aldrich) and PMA (25 ng/mL; Sigma-Aldrich) for 5 h at 37°C, with brefeldin A (10 mg/mL; Sigma-Aldrich) added after 1 h. Then, the cells were fixed and permeabilized with fixation/permeabilization working solution for 20 min at 4°C followed by incubation with monoclonal antibodies against IFN-γ (XMG1.2, BD Biosciences), IL-17A (TC11-18H10.1, BD Biosciences), and IL-10 (JES5-16E3, BD Biosciences). The cells were analyzed using a Cytofix/Cytoperm Plus Kit.