Ly 6g brilliant violet 510

Heart single-cell suspension and flow cytometry was prepared as previously described [30 ]. Briefly, hearts were perfused, extracted, finely minced, and then incubated with digestive enzymes at 37 °C on a rocking shaker at 50 rpm for 45–60 min. Samples were homogenized with a 40 μm cell strainer. Red blood cells were lysed using ammonium-chloride-potassium (ACK) lysis buffer. Next, samples were centrifuged at 400× g for 5 min at 4 °C, and the pellet was then suspended with FACS buffer. Cells were immune-stained with the following anti-mouse antibodies: CD45-Alexa Fluor^®® 700 (BioLegend, 103128, San Diego, CA, USA), CD11b-PerCP (BioLegend, 101228, CA, USA), F480-PE (BioLegend, 123110, CA, USA), CD206-BV421 (BioLegend, 141717, CA, USA), CD64-APC (BioLegend, 161006, CA, USA), Ly-6G- Brilliant Violet 510™ (BioLegend, 127633, CA, USA), Ly-6C-PE/Cyanine7 (BioLegend, 128018, CA, USA), Propidium iodide (Sigma, 25535-16-4, Darmstadt, Germany). Cells were incubated (30 min, 4 °C) with the antibody mixture in a staining buffer (PBS containing 1% bovine serum albumin and 0.05% sodium azide) and then washed twice with a staining buffer. Cells were acquired using a LSRFortessa flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). The data were analyzed using FlowJo V.10 software (FlowJo, Ashland, OR, USA).

Mice were euthanised with CO₂, and blood was collected by cardiac puncture into EDTA-containing tubes (100 mmol/l EDTA). After lysing erythrocytes with RBC lysis buffer (eBiosciences, San Diego, USA) and washing with PBS, antibodies and viability dye were incubated with white blood cells in FACS buffer (PBS with 0.5% BSA and 2 mmol/l EDTA) for 20 min at 4°C. The following antibodies were used (Biolegend, San Diego, USA): CD45-FITC (30-F11 clone), Ly6G-Brilliant Violet 510 (1A8 clone), Ly6C-PE-Cy7 (HK1.4 clone), CD11b-APC (M1/70 clone), F4/80-PE (BM8 clone), CCR2-Brilliant Violet 421 (SA203G11 clone) and CD14-PE-Dazzle 594 (Sa14-2 clone). Viability dye (Fixable Viability Dye eFluor 780, eBiosciences) was added to distinguish live cells. Cells were washed in FACS buffer and fixed for 20 min at 4°C (Perm/Fix buffer, BD Biosciences, USA) before analysis on a Novocyte cytometer (Acea, USA).