Anti cd11b percp cy5.5 clone m1 70

Both retinas from each mouse were dissected, pooled, and digested with 1.5 mg/mL collagenase A (Sigma-Aldrich) for 30 min at 37 °C in a water bath. Digestion was stopped by adding PBS−10% FBS, the retinas were mashed through a 100-µm cell strainer, and the cell suspension was then subjected to flow cytometry staining. To exclude dead cells from the analysis, samples were stained with Ghost Dye 780 (Tonbo Biosciences, San Diego, CA, USA), following the protocol recommended by the manufacturer. Next, the samples were treated with Fc block (obtained from the supernatant of the 2.4G2 hybridoma) prior to surface staining with the following antibodies: anti-CD45-eFluor450 (clone 30-F11, Biolegend, San Diego, CA, USA); anti-CD11b-PerCPCy5.5 (clone M170, Biolegend); anti-F4/80-APC (clone BM8, eBioscience, San Diego, CA, USA), anti-I-A/I-E-PE (clone M5/114.15.2, Biolegend); anti-Ly6c-biotin (clone HK1.4, Beckman Coulter, Indianapolis, IN, USA); and anti-CD11c-FITC (clone HL3BD, BD Pharmingen, San Diego, CA, USA). All samples were incubated with streptavidin-Brilliant Violet 605 (Biolegend) for anti-Ly6c-biotin detection.
Events were acquired using an FACS Canto II cell analyzer (Becton Dickinson, Franklin Lakes, NJ, USA), and all cell doublets and dead cells were excluded from the analysis. Data analysis was performed using FlowJo v10 software (Tree Star, Ashland, OR, USA).