Apc conjugated anti il 17

Eye-draining lymph nodes and conjunctival tissues were collected and single-cell suspensions were prepared. Conjunctival tissues were first digested in RPMI 1640 (Invitrogen) with 2 mg/ml DNase I and 2 mg/ml collagenase type IV (Roche) at 37 °C. The following antibodies were used: FITC-conjugated or Brilliant Violet 421-conjugated anti-CD4 (Biolegend), APC-conjugated anti-IL-17, FITC- conjugated anti-IFN-γ, PE-conjugated anti-RORγt, PE-conjugated anti-T-bet, PE-conjugated anti-CD154 (all from eBioscience), and PE-conjugated anti-IL-23R (R&D System). For intracellular cytokine staining, cells were first stimulated with PMA plus inomycin (Sigma-Aldrich) for 5 h at 37°C and 5% CO₂ in the presence of GolgiStop (BD Biosciences). All the Abs with their matched isotype control and fix-perm buffer were purchased from Thermo Fisher Scientific. Stained cells were examined with an LSR II flow cytometer (BD Biosciences), and the results were analyzed using FlowJo software (Tree Star).