Fluorescence-activated cell sorting (FACS) analysis and detection of cytokine-expressing lymphocytes

The cells were suspended in FACS buffer (7.7 mM NaN3, 2 mM EDTA and 2% FBS dissolved in PBS) to produce a single-cell suspension. Myeloid cells were stained with surface marker antibodies, including BV 421-conjugated anti-CD45 (BD Pharmingen™, San Diego, CA, USA), 7-AAD-conjugated anti-CD11b (BD Pharmingen™), Alexa 647-conjugated anti-F4/80 (eBioscience, San Diego, CA, USA), Alexa 488-conjugated anti-Ly6G (BD Pharmingen™), and Alexa 647-conjugated anti-Ly6C (BioLegend, San Diego, CA, USA). Lymphoid cells were stained with surface marker antibodies including PE-conjugated anti-CD45 (BD Pharmingen™), BV 421-conjugated anti-TCRγδ (BD Pharmingen™), and Alexa 488-conjugated anti-CD4 (BioLegend), in the dark for 30 min at 4 °C. For transcription factor staining, cells were fixed and permeabilized for 30 min at 4 °C using BD Cytofix/Cytoperm buffer (BD Biosciences, La Jolla, CA, USA), washed twice with BD Perm/Wash buffer and then stained with transcription factor marker antibodies, including 7-AAD-conjugated RORγt (BD Pharmingen™) and Alexa 647-conjugated Foxp3 (BioLegend), in the dark for 30 min at 4 °C. For intracellular cytokine staining, cells isolated from the retina were activated with 20 ng/ml PMA (Sigma‒Aldrich), 1 μM ionomycin (Sigma‒Aldrich), and BFA (1:1000, BD Pharmingen™) and cultured for 5 h in 5% CO₂ at 37 °C. Cells were stained with surface marker antibodies including APC-Cy7-conjugated anti-CD45 (BD Pharmingen™) and PE-conjugated anti-CD4 (BD Pharmingen™). Then, the cells were fixed and permeabilized for 30 min at 4 °C using BD Cytofix/Cytoperm buffer (BD Biosciences), washed twice with BD Perm/Wash buffer and then stained with Alexa 488-conjugated anti-IL-17A (BD Pharmingen™) in the dark for 30 min at 4 °C. The cell samples were acquired using a BD FACS Canto II Flow Cytometer (BD Biosciences) using BDFACS DIVA software (BD Biosciences) and analyzed with FlowJo software (v.10, FlowJo™).