Anti γδtcr

Mice ear tissues were cut into pieces and placed in 2.5 mg/ml Dispase II (Roche) solution at 4°C overnight. Then the epidermis was torn off and digested with 0.025% trypsin at 37°C for 15 min, and an equal volume of FBS was added. After being filtered through a 70-μm cell strainer, the cells were resuspended in PBS with 1% FBS. For detecting Tregs, mice ear tissues were cut into pieces and placed in the digestion cocktail (a solution consisting of 2 mg/ml collagenase XI [Cat# C9407; Sigma-Aldrich], 0.5 mg/ml hyaluronidase [Cat# H3506; Sigma-Aldrich], and 0.1 mg/ml DNase [Cat# D5025; Sigma-Aldrich]) with shaking at 255 rpm in 37°C for 40 min (Moreau et al., 2021 (link)). Then an equal volume of FBS was added. After being filtered through a 70-μm cell strainer, the cells were resuspended in PBS with 1% FBS. Dead cells were excluded using Fixable Viability Dye eFluor 780 (eBioscience). The single-cell suspensions were stained per the manufacturer’s recommendations with anti-CD207 (Cat# 144206; BioLegend), anti-MHC II (Cat# 107605; BioLegend), anti-CD3ε (Cat# 152311; BioLegend), anti-γδ TCR (Cat# 118107; BioLegend), anti-CD3 (Cat# 152311; BioLegend), anti-CD4 (Cat# 100406; BioLegend), and anti-Foxp3 (Cat# 563101; BD Pharmingen). After washing with PBS, the cells were subjected to flow cytometry analysis (BD Fortessa II), and the data were analyzed using FlowJo v10.4.

Sorted mouse CD45⁺CD4⁻CD8⁻cells from C57BL/6 WT cultured skin γδ T cell lines were stimulated with rmIL-23 (5ng/ml) or rmIL-1β (10ng/ml) at 37°C for 30min. Cells were fixed with 4% paraformaldehyde and then permeabilized with 0.3% (v/v) Triton X-100. Cells were incubated with anti-γδTCR (Biolegend) and anti-p-Stat3 (Tyr705, Cell Signaling Technology) or anti-p-mTOR (Ser2448, Abcam) at 4 °C overnight followed by donkey anti-rabbit secondary Ab and DAPI for nucleus. Images were acquired with fluorescence microscope (Nikon).