Facscanto 2 cell analyzer

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).

Tumor was dissected and filtered through a cell strainer (100 µm, Corning, USA) in PBS. Red blood cells (RBC) were lysed using ACK (Ammonium-Chloride-Potassium) RBC Lysing Buffer (0.15 M NH₄Cl, 10.0 mM KHCO₃, 0.1 mM Na₂ EDTA), and the B16-F10 and immune infiltrating cells were kept in PBS. One million cells per well were stained in a round bottom 96 well plate using a two-step staining protocol. First, cells were stained with a live/dead dye (Fixable aqua 405 nm, Invitrogen, USA) at 4°C for 20 min, cells were washed, and 100 μL final volume of a solution containing surface antibodies diluted in staining buffer (1% fetal bovine serum, FBS, 1 mM EDTA, and 0.02% NaN₃ in PBS) were added into each well. After 30 min at 4°C, the samples were washed (2X) and resuspended in staining buffer until acquisition. The following antibodies were used: PerCP-Cy5.5 Anti-Mouse CD80 (Clone 16-10A1 Cat no. 194722), APC-Cy7 anti-Mouse F4/80 (Clone BM8 Cat no. 123118), FITC Anti-Mouse CD206 (Clone C068C2 Cat no.141704), from Biolegend, USA, and PE-Cy7 anti-mouse CD86 (Clone GL1 Cat no. 560582) from BD, USA. Samples were assessed with a FACSCanto II cell analyzer (Becton Dickinson, USA) using DiVA 8 acquisition software and FlowJo 5 V10 (Becton Dickinson, USA) data analysis software.

Day 18–20 cardiomyocytes were dissociated using ×1 TrypLE Select (Thermo Fisher Scientific #12563011). For intracellular staining, cells were fixed and stained using the FIX & PERM kit (Thermo Fisher Scientific; #GAS004) according to the manufacturer’s instructions. For cell surface antigens, the antibody was added to the cell suspension resuspended in a buffer containing 10% BSA (Sigma-Aldrich, #A8022) and 0.5 M EDTA (Thermo Fisher Scientific #15575020). All antibody incubations were performed for 30 min on ice protected from light. Acquisition was performed on FacsCanto II Cell Analyzer (Beckton Dickinson). Data was analyzed using FlowJo version 10. Antibody information is provided in the Key resources table.