Figure 7.

Detection of interleukin-10 (IL-10)-producing B cells by ELISPOT and intracellular staining. Purified B cells were cultured unstimulated, stimulated with 10 μg/ml lipopolysaccharide (LPS) or stimulated with LPS in the presence of IL-12, IL-21, interferon (IFN-γ) or transforming growth factor-β (TGF-β). After a 48-hr pre-incubation, the number of IL-10-producing cells was determined by ELISPOT or by flow cytometry. (a) A typical ELISPOT experiment (one of five similar ones) is shown: (i) unstimulated cells, (ii) cells stimulated with LPS, or cells stimulated with LPS in the presence of (iii) IL-12, (iv) IL-21, (v) IFN-γ or (vi) TGF-β. Lanes 1 and 2: 1·0 × 10⁵ cells per well, lanes 3 and 4: 0·5 × 10⁵ cells/well. (b) An average number of IL-10-producing cells as detected by ELISPOT in cultures containing cytokines is demonstrated. Each bar represents the mean ± SD from five independent determinations. Values with asterisks are significantly (*P < 0·01, **P < 0·001 ***P < 0·0001) different from the control (the cells stimulated with LPS in the absence of added cytokines). (c) Representative dot plots demonstrating the percentages of CD19⁺ IL-10⁺ cells, as detected by flow cytometry after intracellular staining. One representative figure from three independent experiments is shown.