Flow cytometry

Single-cell suspensions from the GC tissues, adjacent normal tissues, and blood were washed with FACS-staining buffer (PBS with 0.5% BSA and 0.1% sodium azide) and then incubated with amine-reactive dead cell stain ({"type":"entrez-nucleotide","attrs":{"text":"L34961","term_id":"522204","term_text":"L34961"}}L34961; Thermo Fisher Scientific, Waltham, MA, USA) for 30 min on ice. After another wash, the cells were stained with the following fluorescent-conjugated anti-human surface Abs for 20 min at 4^oC: anti-human CD206 (19.2), anti-human CD11c (B-ly6), anti-human CD86 (IT2.2), anti-human CD15 (W6D3), anti-human CD68 (Y1/82A), anti-human CD66b (WM-53), anti-human CD14 (M5E2), anti-human CD16 (3G8), anti-human CD33 (G10F5), anti-human HLA-DR (L243), anti-human CD45 (HI30), anti-human CD11b (ICRF44), anti-human CD274 (MIH1), anti-human CXCR4 (12G5), anti-human CD56 (NCAM16.2), anti-human CD25 (2A3), anti-human CD19 (HIB19), anti-human CD8a (RPA-T8), anti-human CD4 (RPA-T4), anti-human Foxp3 (206D), anti-human NKP46 (9E2), anti-human PD-1 (EH12.2H7), anti-human IL-17A (BL-168), anti-human IFN-γ (4S.B3), anti-human TNF-α (Mab11), anti-human granzyme B (GB11). To stain intracellular Ags, the cells were first fixed and permeabilized with IC fixation buffer (00-8222-79; eBioscience, San Diego, CA, USA) and 1× permeabilization buffer (00-8333-56; eBioscience). The fixed and permeabilized cells were then stained with fluorescent-conjugated intracellular Abs for 1 hr at room temperature. For nuclear Foxp3 staining, the Foxp3 staining kit was used according to the manufacturer's instructions (00-5523-00; eBioscience). For intracellular cytokine staining, the cells were first stimulated in the presence of brefeldin A (420601; Biolegend) with 50 ng/ml PMA (P1585; Sigma) and 500 ng/ml ionomycin (I0634; Sigma) for 4 h in a 37°C 5% CO₂ incubator. All stained cells were washed and filtered through a 40-μm cell strainer and then analyzed on a LSRFortessa (BD Biosciences) or a FACSymphony A3 (BD Biosciences) flow cytometer.