Murine Retinal Endothelial Cell-T Cell Co-culture

Murine retinal endothelial cells (mREC) were cultured at 80% confluency, as previously described [26 (link),36 (link)]. Spleens were removed, a single-cell suspension was generated, and incubated in erythrocyte lysis buffer (eBioscience) for 5 min at 37 °C, and then washed and counted. CD3⁺ T cells were negatively selected from isolated splenocytes using mouse CD3+ T cell enrichment columns (R&D). Cells were washed, and 1 × 10⁵ T cells of non-diabetic or diabetic C57BL/6 or RORγt^−/− mice were co-cultured with 3 × 10⁵ murine retinal endothelial cells per well. Cells were stained with 10 μg/mL Propidium Iodide, incubated for 48 h, and imaged using a Leica DMI 600B inverted microscope. Additionally, co-cultured cells were then stained with 0.25 μg/tube CD144 (BD Bioscience) for flow cytometry quantification of cell viability, as previously described [36 (link)]. Alternatively, co-cultured cells were incubated with Annexin V (eBioscience) antibodies for apoptosis analysis per manufacturer’s directions.