Single-cell preparations of spleen were obtained as previously described (47 (link)). Splenocytes were stimulated with each type of the CPSs or with the LPS of χ3761 (50 ng/2 × 106 cells) in the presence of Protein Transport Inhibitor Mixture (eBioscience). The cells were then incubated for 8 h at 37 °C with 5% CO2. Before staining, dead cells were excluded using Zombie Red (Biolegend) and mouse FcR were blocked with the rat anti-mouse CD16/CD32 monoclonal antibody (eBioscience). For surface staining, cells were incubated with fluorophore-conjugated antibodies on ice for 30 min. The eBioscience Intracellular Fixation and Permeabilization Buffer Set was used for intracellular staining. Cells were run on a BD LSRFortessa cell analyzer and analyzed with FlowJo software (TreeStar).
For detection of cytokine expression in CD4+ T cells at 21 d after primary immunization, the following antibodies purchased from Biolegend were used, including rat anti-mouse CD3-Alexa Fluor 700, CD4-FITC, IFN-γ–APC/Cy7, APC/Cy7-conjugated rat IgG1κ (isotype), IL-4–Alexa Fluor 647, Alexa Fluor 647-conjugated rat IgG1κ (isotype). Data are presented as described in the figure legends.
For detection of cytokine expression in CD4+ T cells at 21 d after primary immunization, the following antibodies purchased from Biolegend were used, including rat anti-mouse CD3-Alexa Fluor 700, CD4-FITC, IFN-γ–APC/Cy7, APC/Cy7-conjugated rat IgG1κ (isotype), IL-4–Alexa Fluor 647, Alexa Fluor 647-conjugated rat IgG1κ (isotype). Data are presented as described in the figure legends.
