F4 80 percp cy5

Peripheral blood samples were collected into heparinized tubes. The left ventricle tissues of peri-infarct area were collected in cold staining buffer (2% FBS, 0.05% NaN₃ in PBS). For tissue cytometry analysis, single-cell suspensions of tissues were obtained using gentleMACS™ Dissociator (Miltenyi Biotec). Samples were stained with fluorochrome-conjugated antibodies against extracellular or intracellular markers according to the manufacturer’s protocols. The following antibodies were used for extracellular staining: CD11b-FITC (BD Bioscience), F4/80-PerCP/Cy5.5 (eBioscience) and Ly6C-APC (eBioscience). The intracellular fixation and permeabilization kit (eBioscience) was used according to the manufacturer’s protocol for intracellular staining. Cells were then washed with staining buffer and stained with CD206-APC (eBioscience) and iNOS-PE (eBioscience). After incubation with antibodies, blood samples were treated with red blood cell lysis buffer to remove red blood cells and heart tissue samples were filtered through a 70-μM filter. Flow cytometry was performed using FACS Aria flow cytometer (BD Bioscience), and flow cytometry data were analyzed using FlowJo software (TreeStar, Ashland, OR).

FACS was performed as described previously (10 (link)), using FACSAria III (BD, Franklin Lakes, NJ). Live cells were gated using LIVE/DEAD Aqua Stain (cat #L34957; Life Technologies, Grand Island, NY). For the quantification of immune cell frequencies within the gastric mucosae, the cells were stained with the following antibodies: (i) for myeloid cells: CD11b-eFluor 450 (clone M1/70, cat #48-0112-82; eBioscience, San Diego, CA) and Ly6G-PE (clone 1A8, cat. #127607; BioLegend, San Diego, CA); (ii) for T cells: CD4-FITC (clone GK1.5, cat. #11-0041-85, eBioscience) and CD8-PerCpCy5.5 (clone 53-6.7, cat. #45-0081, eBioscience); (iii) for Natural Killer cells: NK1.1-PECy5 (clone PK136, BioLegend); and (iv) for B cells: B220-PE-Cy7 (clone RA3-6B2, cat. #103221, BioLegend) and IgM-PE (clone eB121-15F9, cat. #12-5890, eBioscience).
For the characterization of gastric myeloid immune cell overlap using 7-color FACS, dissociated gastric cells were stained with: (i) LIVE/DEAD Aqua (cat #L34957; Life Technologies), (ii) CD11b-eFluor 450 (clone M1/70, cat #48-0112-82; eBioscience), (iii) Ly6G-PE (clone 1A8, cat. #127607; BioLegend), (iv) Ly6C-APC-Cy7 (clone HK1.4, cat #128025, BioLegend), (v) F4/80-PerCpCy5.5 (clone BM8, cat #45-4801-80, eBioscience), (vi) CD103-APC (clone 2E7, cat #17-1031-82, eBioscience), and (vii) CD11c-PeCy5 (clone N418, cat #15-0114-81, eBioscience).