Cells were isolated from corneas as previously described (21 (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 (clone 30-F11) and Alexa Fluor700-Gr-1 (clone RB6-8C5) (from BioLegend, San Diego, CA); FITC conjugated anti-CD4 (clone RM4–5), PE-conjugated anti-CD8α (clone 53–6.7), PE-Cy7-conjugated anti-CD11c (clone HL3), (all BD PharMingen); eFluor450-conjugated CD11b (clone M1/70) (from eBiosciences, San Diego, CA). Cells were then analyzed on a flow cytometer (FACSAria with FACSDIVA data analysis software; BD Biosciences).
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Corneal Cell Isolation and Analysis
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Other organizations : Saint Louis University
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Variable analysis
independent variables
- Time points for cornea excision (18 and 23 dpi)
- Cell populations isolated from corneas (CD45+, Gr-1+, CD4+, CD8α+, CD11c+, CD11b+)
- Corneas were incubated in PBS-EDTA at 37°C for 15 minutes
- Corneal stroma was digested in 84 U collagenase type 1 for 2 hours at 37°C
- Cell suspensions were filtered through a 40-μm cell strainer and washed
- No positive or negative controls were explicitly mentioned in the protocol.
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