Cd11b pe cf594

After the harvest of cell-free supernatants at 24 h, BMDM were washed and blocked with Fc block (Fc block CD16/CD32, AH Diagnostics). Then, fixable viability dye (FVD 780, AH Diagnostics) and surface staining (MHCII Alexa Fluor 700, F4/80 PE, CD80 eFluor 450, CD86 PE-Cy7, all AH Diagnostics; and CD11b PE-CF594, BD Biosciences) were performed. In addition, internal expression of iNOS Alexa Fluor 488 (AH Diagnostics) and arginase 1 (Arg1) allophycocyanin (R&D Systems) was determined following fixation with 2% paraformaldehyde and permeabilization with Perm buffer (AH Diagnostics). Flow cytometric analysis was performed on a Gallios flow cytometer, and Kaluza software (Beckman Coulter) was used for analysis (Supplemental Fig. 1B–1E).

Mice were perfused transcardially under isoflurane anesthesia with phosphate-buffered saline (PBS) to flush vessels before euthanasia. After euthanasia, eyes were enucleated into ice-cold phosphate-buffered saline (PBS). The corneas, lenses and hyaloid vasculatures were removed and the retinas isolated immediately. To dissociate retinas into single cells, each retina was incubated in 1 ml of papain dissociation solution (Roche Diagnostics GmbH, REF#10108014001, Mannheim, Germany) prepared according to manufacturer’s protocol for 30 min in a 37 °C water bath. Single cell suspensions were washed with FACs buffer on ice and then incubated with primary antibody cocktails for 45 min at 4 °C in the dark. Antibodies used included CD45 Apc-eFluor780 (Invitrogen, Cat#47-0451-82, Waltham, MA, USA), Ly6C Percp/Cy5.5 (Biolegend, Cat#128012, San Diego, CA, USA), Ly6G (BV605 Biolegend, Cat#127639), CD11b PE-CF594 (BD Biosciences, Cat#562287, Franklin Lakes, NJ, USA), CD31 PE (Invitrogen, Cat#2114546) and Fixable viability Dye eFluor506 (Invitrogen, Cat# 65-0866-14). The cells were then washed with FACs buffer, resuspended and analyzed using BD FACSCelesta flow cytometer (BD Biosciences) and FlowJo™ v10.8 Software (BD Life Sciences, Franklin Lakes, NJ, USA). Gating of the different cell populations was performed as previously published [23 (link)].