Corneal Cell Isolation and Flow Cytometry

Cells were isolated from corneas as previously described [18 (link)]. Briefly, corneas were excised at 18 and 23 dpi and incubated in PBS-EDTA at 37°C for 15 minutes at 37°C. Stromas were separated from overlying epithelium and digested in 84 U collagenase type 1 (Sigma-Aldrich, St. Louis, MO) per cornea for 2 hours at 37°C and then were triturated to form a single-cell suspension. Suspensions were filtered through a 40-μm cell strainer cap (BD Labware, Bedford, MA) and washed and then stained. Suspensions were stained with PerCP-conjugated anti-CD45 (30-F11) and Alexa Fluor700-Gr-1 (RB6-8C5) (from BioLegend, San Diego, CA); FITC conjugated anti-CD4 (RM4-5), PE-conjugated anti-CD8α (53–6.7), PE-Cy7-conjugated anti-CD11c (HL3) (all BD PharMingen); eFluor450-conjugated CD11b (M1/70) (from eBiosciences, San Diego, CA). Cells were then analyzed on a flow cytometer (FACSAria with FACSDIVA data analysis software; BD Biosciences).