Choroid-RPE Explant Angiogenesis Assay

Choroid-RPE was dissected from C57BL/6j male and female 3-week-old mice and sectioned into 0.5- to 1-mm pieces as we previously described (Farnoodian et al., 2015 (link)). The 10 pieces per eye were placed into 35−mm culture dish coated with 0.5 ml of Matrigel (10 mg/ml; BD Biosciences) and allowed to harden (30 min at 37^oC). Endothelial cell growth medium (DMEM containing 10% FBS, 2 mmol/L L−glutamine, 2 mmol/L sodium pyruvate, 20 mmol/L HEPES, 1% non-essential amino acids, 100 μg/ml streptomycin, 100 U/ml penicillin, 55 U/ml heparin, and endothelial growth supplement 100 μg/ml (Sigma, St. Louis, MO, United States; E2759) was then added. After 48 h, the explants were fed every other day with the appropriate vehicle, 400 μM caffeine or 10 μM istradefylline. At 8 days, the explants were fixed with 4% paraformaldehyde and photographed (PFA; Electron Microscopy Sciences, Hatfield, PA, United States; 15710). The area of sprouting was quantified as previously described (Farnoodian et al., 2015 (link)).