Collagenase d treatment

To isolate T cells from eyes of uveitic mice, enucleated eyes were first trimmed of external tissue and then the globe was opened along the limbus to remove the lens. The remaining tissue was minced in HL-1 media (Lonza, Walkersville, MD). Cell/tissue separation was accomplished with 1.0 mg/ml collagenase D treatment (Roche, Indianapolis, IN) for 40 minutes at 37°C. After washing, tissue was dispersed through a 40 µm strainer, resuspended in PBS + 2% FBS + 2mM EDTA sorting buffer and then stained. For lymph nodes (LN), tissue was dispersed through a 40 µm strainer and then resuspended in sorting buffer. Cell populations were analyzed using a BD FACS Calibur (Becton Dickinson, San Jose, CA) and MACSQuant Analyzer (Miltenyi Biotec, Auburn, CA) or were sorted on a FACS Aria II (BD) to 99% purity. To determine the number of Tregs present in eyes of mice months after EAU challenge, eye cells were incubated with CD4⁺ isolation beads (Miltenyi Biotec) prior to staining and then passed through a magnetic column that was integrated into a MACSQuant Analyzer. Since the frequency of Tregs at these late time points was expected to be low, utilization of the column permitted the enrichment of the cells before analysis. All data were analyzed using FlowJo (TreeStar, Ashland, OR).

Eyes were enucleated and external tissues and lenses were removed. The remaining tissue was minced in HL-1 media (Lonza, Walkersville, MD) and incubated with 1 mg/ml collagenase D treatment (Roche, Indianapolis, IN) for 45 min at 37°C. Cells were then dispersed by trituration, washed, filtered, and resuspended in RPMI + 10% FBS.