Purification and Hypoxic Stress Assay of Retinal Ganglion Cells

Primary cultured RGCs were purified by two-step immunopanning method as we described previously (Gao et al., 2016 (link)). RGCs were collected and seeded into 96, 24 and 6-well plates pretreated with mouse-laminin (Trevigen Inc., Gaithersburg, MD, USA) and poly-D-lysine (Sigma–Aldrich, St. Louis, MO, USA). Plates were incubated in a humidified incubator with 5% CO₂ at 37° C. RGCs purity was checked by Thy1.1 and Brn 3b immunocytochemical staining (about 85%) (Gao et al., 2016 (link)). Forty-eight hours after seeding, RGCs were incubated with 200 μM cobalt chloride (CoCl₂, Sigma–Aldrich, St. Louis, MO, USA) to induce hypoxia and apoptosis (Kim et al., 2013 (link)), combined with 0, 10, 20, 50, or 100 ng/ml MANF for 24 h, then incubated 12 h, 24 and 48 h under optimal concentration. Half of the RGC medium was replaced with fresh RGC medium every 3 days.