Sl 7f

CNV lesions were induced by laser photocoagulation. Laser radiation was delivered between the major retinal vessels and at equal distances from the optic disc with a diode laser (DC-3000^®; NIDEK, Osaka, Japan) and a slit lamp delivery system (SL-7F; Topcon, Tokyo, Japan). Through a glass cover used as a contact lens, we delivered laser light (200 mW intensity, 170 μm size, 0.02 s duration) to 4 spots per eye for the lesion size study and to 12 spots per eye for the flow cytometry studies. The rupture of Bruch’s membrane for each lesion was evidenced by bubble formation at the time of laser exposure44 (link)45 (link). Each experimental group consisted of 6 or 10 mice. For the analysis of CNV area, 6 mice per group were subjected to laser treatment and used for the analysis. For flow cytometric analysis, cells isolated from 6 mice were used for a single FACS analysis and cells from 10 mice were used for a single RNA isolation protocol. The results from three independent experiments are shown. Thus, a total of 54 wild-type mice and 54 C3^−/− mice were used for the experiments.

CNV lesions were induced by laser photocoagulation as previously described [23 (link)–27 (link)] using a diode laser (DC-3000^®; NIDEK, Osaka, Japan) and a slit lamp delivery system (SL-7F; Topcon, Tokyo, Japan) with a cover slip as a contact lens. Laser photocoagulation (200 mW intensity, 170 μm size, 0.02 s duration) was applied to 4 spots per eye for the lesion size studies and to 12 spots per eye for the flow cytometric studies. Laser irradiation was delivered between the major retinal vessels, 2 to 3 disk diameters from the optic nerve. Bubble formation at the time of photocoagulation was used as an indication of Bruch's membrane rupture. In order to minimize the effects of subjective bias when allocating animals to treatment, a randomization procedure was used. Each treatment group consisted of 5 to 8 mice. For the comparative studies of CNV area, 6 or 8 mice per group were used for preparing the RPE flatmounts. For flow cytometric analysis, cells isolated from 5 to 6 mice were used for a single FACS analysis, and each experiment was performed in triplicate. Thus, a total of 309 wild-type mice, 8 CD4^−/− mice, 8 Rag2^–/–mice and 53 CCR2^−/− mice were used for the experiments.

Sodium fluorescein (Alcon Laboratories) was instilled via a micropipette into the inferior–lateral conjunctival sac (0.6 µL of 0.5% fluorescein dissolved in 4.4 µL of phosphate-buffered saline, per eye). After staining for 15 seconds, mouse eyes were washed once with phosphate-bufferd saline and then examined using a slit-lamp microscope with cobalt blue light (Topcon SL-7F, Tokyo, Japan). Punctate staining was evaluated in a masked fashion, giving a score of 0 to 3 to each cornea: a score of 0 for no punctate staining, a score of 1 when less than one-third of the cornea was stained, a score of 2 when two-thirds or less was stained, and a score of 3 when more than two-thirds were stained.