Type 2 collagenase

First of all, the sample tissue was washed cleanly using PBS, and the NP tissues were segregated by a stereotaxic microscope and trimmed into pieces. NP tissues were digested by incubation with 0.25 mg/mL type II collagenase (Corning, USA) for 6 hours at 37°C. The digest was filtered and centrifuged, and then supernatant was discarded. After inoculating the cells into the culture dish, cells were maintained in DMEM/F12 medium consisting of 15% foetal bovine serum (Corning, USA) and 1% penicillin-streptomycin (Corning, USA) for 3 weeks at 37°C and 5% CO₂. The follow-up experiment was carried out with the third generation cells. The culture medium should be renewed once every three days. Overexpression or knockdown of miR-27a-3p was simulated by miR-27a-3p mimic or inhibitor which was purchased from GenePharma. NP cells were transfected with miR-27a-3p mimic/inhibitor, RASSF5, or empty vector into by Lipofectamine 2000 reagent (Thermo Fisher Scientific, USA).

Isolation of MAECs was performed according to protocols as described previously with some modifications^{57 (link)}. Briefly, eyes from one litter (5 to 6 pups) of OIR-Adora2a^VEC-KO and Adora2a^WTmice at P17 were enucleated and hemisected. The retinas were dissected out and kept in pre-cooling phosphate-buffered saline (PBS) buffer. Retinas (10 to 12 from one litter) were pooled together, rinsed with PBS buffer, quickly minced into small pieces in a 1.5 ml tube using eye scissors, and digested in 8 ml of collagenase type II (Worthington, 2 mg/ml in serum free DMEM, Corning, NY, USA) for 20 min at 37 °C. Following digestion, DMEM with 20% FBS was added and cells were pelleted. The cellular digests then were filtered through 70-μm and 40-μm nylon filters (Corning, NY, USA), centrifuged at 500×g for 5 min at 4 °C to pellet cells, and cells were washed with pre-cooling PBS containing 0.5% bovine serum albumin (BSA). The cells were resuspended in 100 μl pre-cooled PBS containing 0.5% BSA and 2 mM EDTA, and incubated with CD31-MicroBeads for 10 min at 4 °C (Miltenyi Biotec Inc). After affinity binding, CD31-positive cells were obtained via magnetic separation using a MACS separator (Miltenyi Biotec). Purified ECs were immediately lysed for Real-Time PCR assay.