Intrapre reagent

T cells and macrophage was isolated from peripheral blood mononuclear cells by using isolation kit to T cells and monocyte respectively (Miltenyi Biotec). T cells and macrophages plus GC cell lines (MKN45 and SGC-7901 were purchased from ATCC) were maintained in vitro and stimulated by Helicobacter pylori lysate (NCTC 11637, CagA + and VacA+) and analyzed by intracellular cytokine staining. For intracellular cytokine staining, T cells were stimulated at 37°C for 5 hours with a Leukocyte Activation Cocktail (BD Pharmingen). Furthermore, T cells, macrophages and GC cell lines were stained with surface markers, fixed, and permeabilized with IntraPre Reagent (Beckman Coulter), and finally stained with intracellular markers. Data were acquired on FACSVantage SE and analyzed with CellQuest software. Fluorochrome-conjugated mAbs against IL-23A were purchased from BD Pharmingen (Cat. 562468).

Peripheral blood mononuclear cells (PBMCs) were isolated from the fresh blood of patients using Ficoll density gradients as described previously [16 (link)]. Isolated PBMCs were stained for surface markers, fixed, permeabilised with IntraPreReagent (Beckman Coulter, Fullerton, CA) and further stained with antibodies directed against intracellular markers. Leukocytes were stimulated with Leukocyte Activation Cocktail (BD Bioscience, USA) at 37 °C for 4 h prior to intracellular staining using the manufacturer’s staining protocol. Anti-human mAbs against CD3-PE-CF594, CD56-FITC, NKG2D-PE, NKp46-PE-CY7, NKp-30-APC, NKp44-PE, NKG2A-APC, CD69-PE-CY7, PD1-Pacific blue, Tim-3-APC, perforin-APC, Granzyme B-BV421, IFN-γ-PE, and TNF-α-PE with corresponding isotype-matched controls were purchased from BD Biosciences (San Jose, CA, USA). Data were acquired on a Gallios instrument (Beckman Coulter, Brea, CA, USA) and analysed using FlowJo software (Flow jo, LCC, USA).

Peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood samples using Ficoll density gradients as described previously^{34 (link)}. Isolated PBMCs were stained for surface markers, fixed, permeabilized with IntraPreReagent (Beckman Coulter, Fullerton, CA), and further stained with antibodies directed against intracellular markers. Leukocytes were stimulated with Leukocyte Activation Cocktail (BD Bioscience, San Jose, CA, USA) at 37 °C for 4 h prior to the intracellular staining using Pharmingen’s staining protocol. Anti-human mAbs against CD3-PE-CF594, CD56-FITC, IFN-γ-PE, and TNF-α-PE with corresponding isotype-matched controls were purchased from BD Biosciences (San Jose, CA, USA). Data were acquired on a Gallios instrument (Beckman Coulter, Brea, CA, USA) and analysed with FlowJo software (FlowJo, LCC, USA).

Leukocytes were stained with surface markers, fixed, permeabilized with IntraPre Reagent (Beckman Coulter, Fullerton, CA, USA), and further stained with the following antibodies against intracellular markers: anti-CD3 monoclonal antibody (mAb), anti-CD56 mAb, and anti-IFN-γ mAb (BD Biosciences). The FlowJo software program (version 10, FLOWJO, BD Biosciences) was used for data analysis. For the measurement of intracellular cytokine production, cells were stimulated at 37 °C for 5 h with Leukocyte Activation Cocktail (BD Biosciences) before staining, as previously described [29 (link)]. When we checked cell viability after thawing of frozen cells, the viability were good with more than 75%. Cell viabilities were approximately checked 65% using dead cell marker. (Additional file 2: Fig. 2). We analyzed IFN-γ positive NK cell population and the gating process was summarized at Fig. 1A. Finally, T-cell producing IFN-γ was analyzed (Additional file 3: Fig. 3).Fig. 1

The gating process of flow cytometric analysis (A) and IFN-γ production of NK cell with high IFN-γ (+) NK cell, low IFN-γ (+) NK cell, and isotype control (B). FSC-A, forward scatter area; FSC-H, forward scatter height; SSC, side scatter; MNCs, mononuclear cells; CD, cluster of differentiation; NK, natural killer, IFN-γ, interferon gamma

According to the manufacturer’s instructions, peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll density gradients. PBMCs were stimulated with Leukocyte Activation Cocktail (BD Pharmingen, San Diego, CA) at 37 °C for 4 h prior to intracellular staining using the manufacturer’s staining protocol. PBMCs were stained for surface markers, fixed, permeabilized with IntraPreReagent (Beckman Coulter, Fullerton, CA), and then stained with antibodies for intracellular markers. Anti-human mAbs against APC-CD4, FITC-CD56, FITC-IFN-γ, PE-CF594-CD3, PE-IL-2, PE-TNF-α, and V450-CD8, with controls, were purchased from BD Biosciences (San Jose, CA, USA). Data were acquired on a Gallios instrument (Beckman Coulter, Brea, CA) and analyzed with FlowJo software (Ashland, OR).

Human peripheral monocytes and TAMs were isolated using anti-CD14 or anti-CD68 magnetic beads and stimulated with lipopolysaccharide (LPS) for 4 hours, followed by flow cytometry. The mouse macrophages were isolated as described previously20 (link) and treated with LPS for 4 hours, followed by flow cytometry. Cells were stained with surface markers, fixed and permeabilized with IntraPre reagent (Beckman Coulter, Fullerton, CA), and finally stained with intracellular markers. Data were acquired on a FACSVantage SE instrument and analyzed with CellQuest software. The fluorochrome-conjugated antibodies used are described in online supplementary table 3.