Anti f4 80 apc clone bm8

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).

Cells counts were performed using a Z2 Coulter Counter (Beckman Coulter, USA). 3X10⁶ cells from single cell suspensions were incubated at 4 °C for 20 min with the following fluorochrome-conjugated anti-mouse markers: anti-CD45 APC-Cy7 (clone 30-F11; Biolegend, San Diego, USA), anti-CD11b PE-Cy7 (clone M1/70; BD Biosciences), anti-CD11c Pacific Blue (clone N418; Biolegend), anti-Ly6C FITC (clone HK1.4; Biolegend), anti-Ly6G Alexa Fluor 647 (clone 1A8; Biolegend), anti-F4/80 APC (clone BM8; eBioscience), and anti-CD206 (mannose receptor; MR) Alexa Fluor 488 (clone C068C2; Biolegend). Fc receptor block (anti-CD16/32 antibody) was added to all markers cocktails. Intracellular CD206 labelling was performed using a CytoFix/CytoPerm kit (BD Biosciences, USA). After surface receptor labelling, cells were permeabilized and incubated with the marker for 30 min at 4 °C in the dark before being washed twice in 1× Perm/Wash buffer (BD Biosciences) and resuspend in FACS buffer. A BD FACS Canto II flow cytometer (BD Biosciences) was used to acquire data. Data was analysed using FlowLogic FCS analysis software (Inivai Technologies, Melbourne, Australia).