To examine macrophages in the retina and choroid, cells were prepared from mouse eyes. To collect a sufficient number of ocular in ltrating cells, 50 burns were delivered to mouse eyes by laser. After laser injury, eyes were enucleated at different time points (1, 2, 3, 5, and 7days). The anterior segment (cornea, iris, and lens) was excised and the posterior segment of the eye including sclera, choroid, and retina was disrupted with scissors and then shaken in DMEM (plus 10% FBS (Gibco Laboratories), 100U/ml penicillin, 100μg/ml streptomycin) supplemented with 0.5mg/ml Collagenase type D (11088874103, Boehringer Mannheim) at 37°C for 60min. The supernatants were collected and passed through a mesh. After 3 washes, viable cells were obtained. A total of 12 eyes (6 individual pools) were examined per group. The cells were stained with PE anti-mouse CD11b (557397, BD Pharmingen), FITC anti-mouse CD206 (MMR, 123005; BioLegend) and PE-Cy5 anti-mouse CD80 Abs (15-0801-81, eBioscience).
RAW 264.7 cells were stained with PerCP anti-mouse CD11b (101230, BioLegend), PerCP anti-mouse F4/80 (123006, BioLegend), PE anti-mouse CD80 (12-0801, eBioscience), and FITC anti-mouse CD206 (141704, BioLegend).
RAW 264.7 cells were stained with PerCP anti-mouse CD11b (101230, BioLegend), PerCP anti-mouse F4/80 (123006, BioLegend), PE anti-mouse CD80 (12-0801, eBioscience), and FITC anti-mouse CD206 (141704, BioLegend).