Anti mouse tnf α

The following antibodies were used for the present study.
Anti-mouse CD3, clone number 17A2, BioLegend, 05112-40-100, V450;
Anti-mouse CD4, clone number GK1.5, BioLegend, 100434, PerCP/Cyanine5.5;
Anti-mouse CD8, clone number 53-6.7, BioLegend, 100714, APC/Cyanine7;
Anti-mouse CD25, clone number 3C7, BioLegend, 101908, FITC;
Anti-mouse CD69, clone number H1.2F3, BioLegend, 104508, PE;
Anti-mouse CTLA-4, clone number UC10-489, BioLegend, 106305, PE;
Anti-mouse PD-1, clone number 29F.1A12, BioLegend, 135210, APC;
Anti-mouse CD19, clone number 1D3, eBioscience, 12-0193-82, PE;
Anti-mouse CD45, clone number 30F11, BioLegend, 103122, Alexa Fluor 488;
Anti-human CD45, clone number HI30, BioLegend, 304025, PerCP;
Anti-mouse IFNγ, clone number XMG1.2, eBioscience, PE;
Anti-mouse IL-17A, clone number TC11-18H10.1, BioLegend, Alexa Fluor 647;
Anti-mouse IL-13, clone number 13A, BioLegend, PE/Cyanine7;
Anti-mouse Foxp3, clone number MF14, BioLegend, PE;
Anti-mouse TNFα, clone number MP6-XT22, BioLegend, Alexa Fluor 488;
Purified Anti-mouse IFNγ antibody, clone number XMG1.2;
Purified anti-mouse IL-4 antibody, clone number 11B11.

Approximately 2 × 10⁶ cells/well were incubated with respective stimulants and anti-CD49d in 24 well plates at 37 °C, 5% CO_2, 100% humidity for 12 hours. Brefeldin A (10 µg/ml) (BD Biosciences, Japan) was added after the first 7 hours followed by a further 5 hours incubation^{66 (link)}. Cells were then washed with staining buffer (SB - 2% FCS, 0.05% NaN3 in PBS) twice. To reduce background, cells were treated with FcR block Ab (2µl/1ml in SB) (BD Biosciences) for 5 min at 4 ℃. They were then washed twice with PBS then stained with Fixable viability dye (FVD) eFlour 506 (1 µl/ml) (Invitrogen) for 30min at 4 ℃. Next was two washing steps with SB, then surface staining in 100 µl SB using anti-mouse CD3 (2 µl/100µl), anti-mouse CD4 (1 µl/100µl), anti-mouse CD8 (1 µl/100µl) (Tonbo biosciences) incubated for 30 min at 4 ℃. Surface-stained cells were then fixed and permeabilized using the Fixation/ Permeabilization solution (BD Biosciences) following the manufacturer’s instructions followed by intracellular cytokine staining with anti-mouse IFN-γ (1 µl/100µl) (Invitrogen eBiosciences), anti-mouse IL2 (1 µl/100µl) (Invitrogen eBiosciences) and anti-mouse TNF-α (1 µl/100µl) (Biolegend)^{67 (link)}. Cells were analyzed using the Acea NovoCyte 3000 flow cytometer and gated using the NovoExpress 1.3.0 software.

From 6 to 16 weeks of age, female KCASP1Tg and WT littermate mice were treated with 10 µg/body of anti-mouse TNF-α (BioLegend, San Diego, CA, USA) or IL-1α/β monoclonal antibody (BioLegend) three times per week intraperitoneally (n = 4, respectively). Phosphate-buffered saline (PBS) was injected as a control (n = 5). All the mice were sacrificed at the age of 16 weeks, and the gastrointestinal tract was investigated.