After OCT imaging, the animal was euthanized and both right (injected) and left (fellow, uninjected) eyes were enucleated and intraocular contents collected separately as described by John et al.
14 Briefly, each eye was rinsed in a flow buffer, placed in 50 µL of flow buffer, the cornea and lens removed, and the aqueous humor, iris, vitreous, retina, and retinal pigmented epithelium were collected. The intraocular contents were incubated with 10 mg/mL of DNAse (Roche, Germany) and 0.5 mg/mL of collagenase (Roche, Germany) at 37°C for 25 minutes, and passed through a 70 µg filter and washed with flow buffer. Single cells were counted using a Nexcelom Cellometer Auto 2000 (Nexcelom Bioscience, MA). For each sample, 1 × 10
6 cells were stained with Zombie Aqua Fixable Viability dye (Biolegend, San Diego, CA) and incubated in Fc Block (2% per sample; Biolegend). The cells were stained with primary antibodies from Biolegend (Lys6G-AF647 [1A8,127609], Lys6C-FITC [HK1.4,128005], CD11b-PerCPCy5.5 [m1/70,101227], CD3-BV421 [17A2, 100228], CD19-BV605 [6D5, 115539], CD11c-APC/fire F780 [N418, 117351], NK1.1-PE [Pk136, 108707], and CD45-BUV395 [30-F11, 565967]) for 30 minutes at 4°C. After staining, cells were washed and fixed with 1% paraformaldehyde in PBS. Data were acquired with a BD LSRII flow cytometer using BD FACSDiva software (BD Bioscience, Franklin Lakes, NJ). Cell counts were obtained using CountBright counting beads according to manufacturer protocols. Compensation was performed using single color controls prepared from BD Comp Beads (BD Biosciences). Cell lineages were defined by the following combination of cell surface markers: T cells (CD45+,CD3+,CD19-), B cells (CD45+,CD3-,CD19+), NK cells (CD45+, CD11b
lo-mid, NK1.1+), NK T cells (CD45+,CD11b
lo-mid, NK1.1+, CD3+), dendritic cells (CD45+, CD11b
hi,CD11c
hi), neutrophils (CD45+, CD11b
hi, Ly6G+), inflammatory macrophages (CD45+,CD11b
hi, Lys6C
hi), microglia (CD45+, CD11b
hi, NK1.1- cd11c-, Ly6c-). The gating strategy is shown in
Supplemental Fig. S2. Data analysis was performed using FlowJo v10.1 software (FlowJo LLC, Ashland, OR).