Cd86 apc 24f

Lymph nodes and spleen tissues were collected after mice were euthanatized on day 30. Single cells were obtained by grinding tissues with 70 μm nylon filters in 0.5% BSA-PBS. Anti-mouse antibodies against CD3-Percp/Cy5.5 (145-2C11, BioLegend), CD4-APC (GK1.5, BioLegend), CD4-FITC (GK1.5, BioLegend), CD8-PE (53-6.7, BioLegend), CD11c-FITC (N418, BioLegend), CD11b-Percp/Cy5.5 (M1/70, BioLegend), CD80-PE (16-10A1, eBioscience), CD86-APC (24F, BioLegend), CD69-FITC (H1.2F3, BioLegend), B220-Percp/Cy5.5 (RA3-6B2, BioLegend), CD138-APC (281-2, BioLegend) or I-A/I-E (MHC II)-PE/Cy7 (M5/114.15.2, BioLegend) were used for cell surface antigen staining. After washing with PBS for three times, cells were treated with Foxp3/transcription factor fixation/permeabilization concentrate and diluent (eBioscience, USA) and incubated with anti-mouse antibodies against IFNγ-PE/Cy7 (XMG1.2, BioLegend) or Ki67-FITC (SolA15, BioLegend) for intracellular antigen staining. Besides, S1 protein with His tag (Sino Biological, China) and anti-His tag-PE (BioLegend) were used to label S1 specific B cells. The cells were analyzed by flow cytometry.

The legs of C57BL/6 male mice (6-8 weeks old) were separated. The bone marrow was flushed out with RPMI-1640 by 1 mL syringe and centrifuged at 1500 rpm for 3 min. Then cells were cultured in RPMI-1640 containing 10% FBS, 1% penicillin-streptomycin and 20 ng/mL granulocyte macrophage colony-stimulating factor (GM-CSF) (PeproTech, UK) with 5% CO₂ at 37°C. Half of the medium was removed on day 3 and an additional culture medium with GM-CSF (20 ng/mL) was added. BMDCs were harvested on day 6-8 and transferred to a 12-well plate at a density of 2 × 10⁵ cells per well. Cells were treated with SMVs (20 μg), OMVs (2 μg), and HMVs (20 μg) with equivalent protein for 24 h, respectively. PBS-treated cells were used as blank control. Cells were then collected, washed, and blocked with anti-mouse CD16/32 (BioLegend). For specific labeling, cells were stained with anti-mouse antibody against CD11c-FITC (N418, BioLegend), CD11b-Percp/Cy5.5 (M1/70, BioLegend), CD80-PE (16-10A1, eBioscience), CD86-APC (24F, BioLegend), H-2K^b/H-2D^b (MHC-I)-PE/Cy7 (28-8-6, BioLegend) and I-A/I-E (MHC-II) -PE/Cy7 (M5/114.15.2, BioLegend) in 0.5% bovine serum albumin (BSA) dissolved in PBS on ice for 40 min. After washing with PBS for 3 times, cells were analyzed by flow cytometry.