Pe anti mouse cd3

For cell-surface labeling, cells were washed with cold PBS and incubated with a blocking antibody for 10 min. Fluorochrome-conjugated antibodies were diluted 1:100 in PBS and incubated with cells on ice for 20 min.
For subcellular labeling, cells were stained using Fix&Perm kit (Multiscence, GAS005/2). 2x10⁵ cells were fixed with 100μL medium A for 15min. After washing with PBS containing 5% FBS, cells were permeabilized with 100μL medium B and stained with IL-6, CCL3, TNFα for 20min respectively. Stained cells were washed with PBS and analyzed on Cytek Aurora instrument.
Antibodies for flow cytometry were as follows: anti-mouse CD45-violetFluor450 (Multi Sciences, AM04512), anti-mouse CD45-PECY7 (BioLegend, 103114), anti-mouse CD19-FITC (Multi Sciences, AM01901), anti-mouse CD3-PE (BioLegend, 100205), anti-mouse CD11b-Percp-cy5.5 (Multi Sciences, AH011B07), anti-mouse GR-1-PE (Multi Sciences, AM0L604), Lamp1-FITC (ProteinTech, FITC-65050), IL-6-eFluor450(ebioscience, 48-7061-82), CCL3-PE(ebioscience, 12-7532-82), TNFα-APC(ebioscience, 17-7321-82).

The harvested tumors of the mice were cut into pieces in DMEM medium containing 2% FBS on ice and then treated with 5 volume equivalent trypsin at 37℃ for 60 min. The samples were filtered and centrifuged at 600 g for 5 min. The cells were washed with PBS containing 2% FBS and co-incubated with sterile ZombieNIR dye and Fc receptor blocker at 4℃ for 15 min. Next, the cells were stained with the primary antibodies in dark for 30 min, including anti-mouse CD86-PE antibody (B334834, Biolegend), anti-mouse CD206-APC (B354282, Biolegend), anti-mouse F4/80-PE/Cyanine-7 (B342137, Biolegend), anti-mouse CD45-Brilliant Violet 421 (B343559, Biolegend), anti-mouse CD3-PE (B341466, Biolegend), anti-mouse CD4-FITC (B269033, Biolegend), anti-mouse CD8a-APC (B348048, Biolegend), anti-mouse CD11c-APC (B339313, Biolegend), anti-mouse I-A/I-E (MHC II)-PE/Cyanine-7 (B337001, Biolegend) and anti-mouse CD11b-FITC (B349919, Biolegend). After rinsing excess antibodies, the cells were immediately assessed by a flow cytometer (Beckman Coulter, USA).

1 × 10⁶ single suspension cells were stained with the proper antibodies diluted in cell staining buffer (420201, BioLegend, USA) for 30 min at 4 °C. For surface staining, anti-mouse CD45-Alexa Fluor^® 700 (Biolegend, 109821), anti-mouse CD11b-PercpCy5.5 (Biolegend, 101227), anti-mouse CD3-PE (Biolegend, 100206), anti-mouse CD3-FITC (Biolegend, 100203), anti-mouse CD4-APC (Biolegend, 100412), anti-mouse CD8-PercpCy5.5 (Biolegend, 100721) and anti-mouse NK1.1-APC (eBioscience, 17-55941-82) were used for extracellular staining. For intracellular staining, the cells were stimulated with or without various cytokines and GolgiPlug for 4 h and then fixed/permeabilized with BD Cytofix/Cytoperm kit as the recommendation of the manufacturer. After washing with wash buffer, anti-human IFN-γ-Percp-cy5.5 (Invitrogen, 45-7319-42), anti-human and Granzyme B-PE (Invitrogen, 12-8899-41) were used for intracellular staining. All samples were determined using FACSalibur flow cytometer (Becton-Dickinson, FL, NJ, USA), and the percentages of cells within each phase of the cell cycle were analyzed using FACS Express 7 software (De Novo Software).

Freshly isolated hepatic lymphocytes separated from liver tissue sections or human PBMCs were stained with various antibodies in the dark at 4°C for 30 min, then washed twice with PBS. After fixation and permeabilization with a Cytofix/Cytoperm kit (BD Biosciences, San Jose, CA, USA) and staining with various antibodies, including anti-mouse CD3-PE (100206, BioLegend), anti-mouse NK1.1-FITC (108706, BioLegend), anti-mouse CD8a-FITC (2002714, Invitrogen), anti-mouse CD4-PE-Cy7 (25-0042-82, eBioscience), anti-mouse IFN-γ-APC (505810, BioLegend), anti-human CD4-PE-Cy7 (25-0049-42, eBioscience), anti-human CD8a-FITC (2518338, Invitrogen), anti-human IFN-γ-APC (17-7319-82, eBioscience). The cells were analyzed by flow cytometry on a BD FACSVerse^TM Flow Cytometer (BD Bioscience) and the analysis was used by The Guava Soft software.

Age- and sex-matched adult mice were used in all flow cytometry experiments. We obtained peripheral blood by cardiac puncture and collected blood into a 1.5-mL Eppendorf tube containing 4% citrate solution. Next, we added the blood-citrate mixture to 3 mL of 2% dextran/1× PBS solution and incubated for 30 min at 37 °C. The upper layer was transferred to a new 5-mL polystyrene FACS tube (Falcon, #352058) and centrifuged at 1200 rpm for 5 min at 4 °C. We then lysed red blood cells for 15 min at room temperature using BD Pharm Lyse (BD, 555899). Cells were washed twice with staining media (Hank’s Balanced Salt Solution (HBSS) containing 2% FBS and 2 mM EDTA). The following antibodies were added (1:100) to each sample and incubated for 30 min at room temperature: Alexa Fluor 700 anti-mouse CD8a (Biolegend, 100730), PE/Dazzle-594 anti-mouse CD4 (Biolegend, 100456), APC anti-mouse CD19 (Biolegend, 115512), Alexa Fluor 488 anti-mouse NK-1.1 (Biolegend, 108718), and PE anti-mouse CD3 (Biolegend, 100205), and Zombie Aqua Fixable Viability Kit (Biolegend, 423101) was used as a live-dead stain. We washed samples twice with staining media and sorted directly into TRIzol LS using a BD Aria FACS.

Spleen and lymph nodes were harvested from immunized mice five days after the final boost. B cells were enriched using theEasySep mouse pan-B cell isolation kit (Stemcell Technologies, Vancouver, CA). Cells were then stained with a fluorophore-labeled antibody panel, which includes PerCP-Cy5.5 conjugated anti-mouse IgM (BD Biosciences), PerCP-Cy5.5 conjugated anti-mouse IgD (BD Biosciences), APC-Cy7 conjugated anti-mouse CD19 (BD Biosciences), and a cocktail of phycoerythrin (PE) conjugated antibodies used during sorting as a dump channel: anti-mouse Ly6g (Biolegend, San Diego, CA, USA), PE anti-mouse CD3 (Biolegend), and PE anti-mouse F4/80 (Biolegend) and Alexa647 and Alexa488 ovalbumin (InvivoGen, San Diego, CA, USA). Single dump channel-/CD19+/IgM-/ovalbumin^duel+-stained B cells were FACS sorted and barcoded using the Chromium controller (10× Genomics, Pleasanton, CA, USA) and a sequencing library was prepared according to the manufacturer’s instructions using the Chromium Next GEM single-cell 5′GEM, BCR amplification and library construction kit (10× Genomics, Pleasanton, CA, USA).