Ic1935g

The conjunctival tissue was separated and subsequently digested in DMEM media containing 2 mg/mL collagenase D (11088866001, Roche, Indianapolis, IN, USA) and 0.2 mg/mL DNase I (10104159001, Roche) for 2 h at 37 °C. The cornea and iris were isolated in RPMI with 2.5 mg/ml Liberase TL (Sigma-Aldrich, St.Louis, MO, USA) and incubated at 37 °C on a shaker for an hour. Tissue suspension was filtered through a 70-µm cell strainer (BD Falcon; Becton-Dickinson, Franklin Lakes, NJ, USA). After washing with 5% FBS, single cells in suspension were incubated with Fc blocking antibody at 4 °C for 30 min. Cells were then immunostained with the APC/Cy7-conjugated anti-CD45 antibody (1:100 dilution, 103116, BioLegend), or isotype-matched control antibody (1:100 dilution, 400624, BioLegend). The Foxp3/transcription factor staining buffer set (00-5523-00, eBioscience), HIF-1α (1:200 dilution, IC1935G, R&D systems, Minneapolis, MN) or isotype-matched control antibody (1:100 dilution, 400132, BioLegend) were used for HIF-1^α staining. Live and dead cells were stained using Zombie Green Fixable viability kit (423111, BioLegend). The stained cells were analyzed using the LSRII flow cytometer (BD Biosciences, San Jose, CA, USA) and FCS Express software (De Novo Software, Los Angeles, CA, USA). The gating strategy is described in Supplementary Fig. S5.

A single-cell suspension of corneal cells was prepared by digesting the individual cornea with Liberase TL (2.5 mg/ml) (Sigma-Aldrich) as previously reported (Gaddipati et al., 2015 (link)). Cells were stained with fluorochrome-conjugated monoclonal antibodies against CD45 (103116, Biolegend), HIF-1α (IC1935G, R&D system) and their isotype controls (400623, 400132, Biolegend). The stained cells were analyzed using the LSRII flow cytometer (BD Biosciences, San Jose, CA) and FlowJo software.