Multicolor Flow Cytometry Analysis of Gastric Immune Cells

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).