Moflo facs sorter

Retina and secondary lymphoid organs (SLO, including draining LN and spleens) from CAU mice (week 12 – 16 post-induction) were harvested and pooled, and CD4⁺ T cells were isolated via negative selection with a magnetic CD4⁺ T cell isolation kit (Cat #: 130-104-454, Miltenyi Biotec Inc.). The isolated cells were >98% CD4⁺ as confirmed by flow cytometry, and they were stained with FITC- or PerCP/Cy5.5-anti-CD44 (clone IM7, Cat #: 103006 or 103032, BioLegend) antibody, and sorted for CD44^hi memory and CD44^−~lo control subpopulations using a MoFlo^® FACS sorter (Dako Cytomation) or a BD FACSAria^™ III sorter (BD Biosciences). The sorted cells in equal live numbers (1×10⁵) were cultured for 5 days and stained with 7-AAD (Cat #: 00-6993-50, ThermoFisher), and the viable cells were determined as 7-AAD unstained populations by flow cytometric analysis.

Draining lymph nodes from DED mice were harvested and pooled, and CD4⁺ T cells were isolated via negative selection with a CD4⁺ T cell isolation kit (Miltenyi Biotec Inc.). The isolated cells were >98% CD4⁺ as confirmed by flow cytometry, and they were stained with FITC-anti-CD44 (clone IM7) and PE-anti-CD62L (clone MEL-14, BioLegend) antibodies, and sorted for CD44^loCD62L^- effector subpopulation using a MoFlo^® FACS sorter (Dako Cytomation). The sorted CD44^loCD62L^-CD4⁺ cells were treated with IL-23 (10 ng/ml, eBioscience), IL-2 (50 IU/ml, Pepro Tech), IL-23+IL-2, monoclonal CD25 blocking Ab (CD25 mAb, 10 μg/ml, clone PC61.5, eBioscience), or IL-23+CD25 mAb for 48 hours. Memory Th17 cells, as well as expression levels of IL-7R, IL-15R, and Annexin V were then examined by flow cytometry.