Anti mouse cd3 fitc

Mouse CD8⁺ T cells were stained with antigen-specific tetramers as described previously [11 (link)]. Briefly, AFP-, GPC3- and OVA-specific tetramers were generated as per manufacturer’s instructions (the QuickSwitchTM Quant H-2K^b tetramer kit, TB-7400-K1, MBL, USA) with AFP212 (GSMLNEHVM) [13 (link)], GPC3 (AMFKNNYPSL) [31 (link)] and OVA (SIINFEKL) [28 (link)] (10 μM) epitopes, respectively. Lymphocytes derived from treated mice were treated with 50 nM dasatinib (HY-10181, MCE, USA) for 30 min at 37 °C, followed by washing with 1X washing buffer and stained with AFP-H-2K^b-tetramer-PE, GPC3-H-2K^b-tetramer-PE or OVA-H-2K^b-tetramer-PE (2 μg/ml) for 60 min at 4 °C. Subsequently, tetramer-stained lymphocytes were counterstained with APC-cy7-CD45 (eBioscience, USA), FITC-anti-mouse CD3 (eBioscience, USA) and APC-anti-mouse CD8α (BioLegend, USA) antibodies. Stained cells were subjected to flow cytometric analysis with BD LSRFortessa (BD, USA) and analyzed by FlowJo software (FlowJo, LLC).

Cells were incubated with Fc block antibody (#14-0161-82, eBioscience, 1:100) for 30 min at 4 °C to prevent nonspecific binding. Cell surface labeling was conducted with fluorescently conjugated antibodies for 30 min at 4°C. The following antibodies were used: BV510 anti-mouse CD45 antibody (#103137, Biolegend, 1:25), PerCP anti-mouse CD11b (#101229, Biolegend, 1:50), APC anti-mouse F4/80 (#17-4801-80, eBioscience, 1:25), PE/Cy7 anti-mouse Gr1 (#108415, Biolegend, 1:100), FITC anti-mouse CD3 (#11-0032-82, eBioscience, 1:50), PE/Cy7 anti-mouse CD19 (#25-0193-81, eBioscience, 1:50), BV421 anti-mouse NK1.1 (#108741, Biolegend, 1:20), PE anti-mouse CD36 (#562702, BD Biosciences, 1:100), PE anti-mouse CD4 (#4329629, Invitrogen, 1:200), APC anti-mouse CD8a (#17-0081-81, eBioscience, 1:200), BV421 anti-mouse CD206 (#141717, Biolegend, 1:25), FITC anti-mouse CD80 (#FITC-65076, Proteintech, 1:200), FITC anti-mouse GzmB (#372206, Biolegend, 1:25), FE anti-mouse IFNγ (#PE-65153, Proteintech, 1:50). Fluorescence data were collected using a FACSAria II flow cytometer (BD Biosciences, USA) and analyzed employing a FlowJo software. The single cell population was separated with a BD FACSAria II Cell Sorter.

For cell surface marker staining, cells were stained with antibody on ice for 30 min. For intracellular staining, cells were fixed and permeabilized by Intracellular Fixation & Permeabilization Buffer Set (Thermofisher Scientific, USA) for 30 min, followed by antibody incubation recommended concentration for 30 min. Finally, data was acquired using Apogee flow system (A60‐Universal, UK) and FlowJo_v10.8.1 software. The following fluorochrome‐labeled antibodies and staining kits were used for flow cytometry according to the manufacturers’ protocols: anti‐mouse‐CD3 FITC (45‐0031‐82, Thermofisher Scientific, USA), anti‐mouse‐CD3 PerCP‐Cyanine5.5 (11‐0031‐82, Thermofisher Scientific), anti‐mouse‐CD4 PE (12‐0041‐82, Thermofisher Scientific), anti‐mouse‐ IFN‐γ FITC (Cat# 11‐7311‐82, Thermofisher scientific, USA), anti‐mouse‐ IL‐4 APC (Cat# 17‐7041‐82, Thermofisher Scientific), anti‐mouse‐ IL‐17A PerCP‐Cyanine5.5 (12‐0041‐82, Thermofisher Scientific), mouse regulatory T cell staining kit (88–8111, Thermofisher Scientific), Rat IgG2a kappa Isotype Control (eBR2a) PerCP‐Cyanine5.5 (45‐4321‐80, Thermofisher Scientific), Rat IgG1 kappa Isotype Control (eBRG1) APC (17‐4301‐81, Thermofisher Scientific), Rat IgG1 kappa Isotype Control (eBRG1) FITC (11‐4301‐81, Thermofisher Scientific), Rat IgG2a Anti‐Mouse control PE (88‐8111‐40, Thermofisher Scientific).

Nicotine was purchased from Sigma-Aldrich (St. Louis, MO, USA). Monoclonal antibodies (anti-mouse CD3-FITC, anti-mouse CD4-APC, anti-mouse CD8-PE-cy7, Rat IgG2b K Isotype Control FITC, Rat IgG2b K Isotype Control APC and Rat IgG2a K Isotype Control PE-cy7) and Annexin V PE Apoptosis Detection kit were purchased from eBioscience (San Diego, USA). Anti-mouse CD95-PE-cy7 was obtained from BD Biosciences (New Jersey, USA). Mouse IgG1 and IgG2a ELISA kits were obtained from MultiSciences (Hangzhou, Zhejiang, China). Mouse IL-4 ELISA kits were obtained from Dakewe Biotech (Shenzhen, Guangdong, China). Trizol was purchased from Life Technologies (Gaithersburg, MD, USA). Reverse transcription and RT-qPCR kits were purchased from TaKaRa Biotechnology (Dalian, Liaoning, China). All primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). All chemicals and reagents were analytical grade.

OVA, LPS and Andrographolide were purchased from Sigma-Aldrich (St. Louis, MO). Water-soluble Andrographolide sulfonate (Xi-Yan-Ping Injection) was provided by Jiangxi Qingfeng Pharmaceutical Co., Ltd. ELISA kits for TNF-α, IL-6, IL-4 and IL-1β were purchased from Dakewei (Beijing, China). Anti-phosphorylation of p65 and anti-p-p65 were purchased from Cell Signaling Technology (Beverly, MA). Anti-NLRP3 and anti-CASP1 (3345-1) were purchased from Epitomics. Anti-ASC and anti-Actin (sc-1616) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-mouse CD3-FITC, CD11b-PE and CD11c-APC antibodies were bought from eBioscience. GTVisin™ anti-mouse/anti-rabbit immunohistochemical analysis KIT was purchased from Gene Company (Shanghai, China). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO).