Bv421 anti tcrγδ

The eyeball blood of mice was collected with EDTA anticoagulant tube on the 14^th and 21^st day after operation. Red blood cells were lysed by lysing buffer (BD Pharmingen, CA, USA) according to the manufacturer’s protocol. For cell surface staining, aliquots of single cell suspensions (1×10⁶) were incubated with fluorophore-conjugated monoclonal antibodies at room temperature in the dark (Alexa Fluor 488-anti-CD3, PE-cy7-anti-CD4 or EF450-anti-CD4, PE-cy5.5-anti-CD8, BV421-anti-TCRγδ and/or APC-anti-CD25; all from Biolegend, San Jose, CA, USA). For intracellular staining, cells were stimulated by incubation for 4 h in RPMI 1640 medium, phorbol 12-myristate 13-acetate (50 ng/mL), ionomycin (1 µg/mL), and brefeldin A (1 µg/mL) (all from Biogems, Rocky Hill, NJ, USA) in a 5% CO₂ atmosphere at 37 ℃. The cells were then washed with PBS, fixed, permeabilized, and stained with PE-cy7-anti-IL-17A (Biolegend, San Jose, CA, USA) according to the manufacturer’s protocol. For intranuclear transcription factor detect, cells were washed, fixed, permeabilized, and stained with PE-anti-Foxp3 (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s protocol. Fluorescence data were collected on a FACS Aria Ⅲ (BD Biosciences, San Jose, CA, USA) and analyzed with FlowJo software (Tree Star, Ashland, OR, US).