24-well plate coated with anti-cd3  (Thermo Fisher)

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Exopolysaccharide from Bacillus subtilis induces anti-inflammatory M2 macrophages that prevent T cell-mediated disease

doi: 10.4049/jimmunol.1601641

Figure Lengend Snippet: CTV-labeled spleen cells were stimulated with anti-CD3 and cultured with F4/80+CD11b+ cells from EPS- or ΔEPS-treated mice. A. (Left): Proliferation of CD4+ T cells; Horizontal bar = % proliferating cells; (center) % proliferating CD4+ T cells; and (right) % activated CD4+ T cells (CD44+CD25+) in 3 independent experiments; N = 7 mice total per group. B. (Left): Proliferation of CD8+ T cells; Horizontal bar = % proliferating cells; (center) % proliferating CD8+ T cells; and (right) % activated CD8+ T cells (CD44+CD25+) in 3 independent experiments; N = 7 mice total per group. C. Proliferation of CD4+ T cells co-cultured with peritoneal CD3+ T cells (Left) or IgM+ B cells from EPS- or ΔEPS-treated mice. Horizontal bar = % proliferation. D. Proliferation of CD4+ T cells cultured with peritoneal cells from EPS-treated TLR4-/ or EPS-treated WT mice. Horizontal bar = % proliferating cells. E. (Left) % proliferating CD4+ T cells in co-cultures with peritoneal cells from TLR4−/− mice and (right) myeloid (mye) MyD88−/− mice in 4 independent experiments; none = splenocytes alone. N = 9 mice total per group. F. Proliferation of CD4+ T cells cultured with peritoneal cells from mice gavaged with WT or epsH B. subtilis spores. (Left) Horizontal bar = % proliferating cells; (right) % proliferating CD4+ T cells in 3 independent experiments; none = splenocytes alone. N = 5 mice total per group.

Article Snippet: For transwell experiments, 1×10 6 splenocytes were cultured in anti-CD3-coated 24-well plates with or without transwell inserts (Corning) containing 5×10 5 peritoneal cells.

Techniques: Labeling, Cell Culture

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Exopolysaccharide from Bacillus subtilis induces anti-inflammatory M2 macrophages that prevent T cell-mediated disease

doi: 10.4049/jimmunol.1601641

Figure Lengend Snippet: A. (Left) Quantification of IL-2 by ELISA of PMA- and ionomycin-stimulated cells from the MLN of C. rodentium-infected mice either untreated, or treated with EPS. (Right) Quantification by of IL-2 by ELISA of PMA- and ionomycin-stimulated MLN cells from C. rodentium-infected mice that received adoptive transfer of F4/80+ or F4/80 peritoneal cells from EPS-treated mice. Data are representative of 2 independent experiments with N = 7–10 mice total per group. B. Quantification by ELISA of serum TNFα, IFN-γ and IL-2 in EPS-treated or untreated (NT) mice 2 h post-i.p. injection of anti-CD3. Average of 3 independent experiments with N = 8 mice total per group. C. Quantification by ELISA of IFNγ, IL-17A, and IL-13 in culture supernatants of anti-CD3-stimulated splenocytes from EPS- or ΔEPS-treated mice. Average of 4 independent experiments with N = 8 mice total per group.

Article Snippet: For transwell experiments, 1×10 6 splenocytes were cultured in anti-CD3-coated 24-well plates with or without transwell inserts (Corning) containing 5×10 5 peritoneal cells.

Techniques: Enzyme-linked Immunosorbent Assay, Infection, Adoptive Transfer Assay, Injection

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Exopolysaccharide from Bacillus subtilis induces anti-inflammatory M2 macrophages that prevent T cell-mediated disease

doi: 10.4049/jimmunol.1601641

Figure Lengend Snippet: A & B Proliferation of CD4+ (A) or CD8+ (B) T cells co-cultured with peritoneal cells from untreated (NT)- or EPS-treated mice in direct contact (no transwell, left) or with peritoneal cells in a transwell insert (right). Horizontal bar = % proliferation. Representative of three independent experiments. C. Proliferation of CD4+ T cells of anti-CD3 stimulated splenocytes cultured with peritoneal cells from EPS-treated mice in the presence of neutralizing anti-TGF-β antibody or mouse (Ms) IgG1 isotype control. Horizontal bar = % proliferation. Representative of three independent experiments. D. % Proliferation of CD4+ T cells cultured with peritoneal cells from EPS-treated mice and containing inhibitors of M2 macrophage function. (■) NT peritoneal cells alone (negative control); (☐) peritoneal cells from EPS-treated mice (positive control); () cultures with inhibitors of M2 macrophages, anti-TGF-β, Nor-NOHA, L-Arginine, NS-398, anti-PD-L2, anti-PD-L1, and anti-IL-10. Data are from 3 independent experiments each, N = 6 mice total per group. E. Quantification by ELISA of total TGF-β in serum 3 days after EPS-treatment or no treatment (NT). N= 4–5 mice total per group. F. % Proliferation of CD8+ T cells cultured with peritoneal cells from EPS-treated mice containing inhibitors of M2 macrophage function. (■) NT peritoneal cells alone (negative control): (☐) peritoneal cells from EPS-treated mice (positive control); () Cultures with inhibitors of M2 macrophages, as in D. D & F. Statistical significance determined by one-way ANOVA in combination with Bonferroni’s test for multiple comparisons. P < 0.0001 by ANOVA for D & F.

Article Snippet: For transwell experiments, 1×10 6 splenocytes were cultured in anti-CD3-coated 24-well plates with or without transwell inserts (Corning) containing 5×10 5 peritoneal cells.

Techniques: Cell Culture, Negative Control, Positive Control, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Exopolysaccharide from Bacillus subtilis induces anti-inflammatory M2 macrophages that prevent T cell-mediated disease

doi: 10.4049/jimmunol.1601641

Figure Lengend Snippet: A. Representative example of Foxp3 expression in CD4+CD25+ T cells in the peritoneal cavity of untreated (NT, top) or EPS-treated (EPS, bottom) mice. B. % CD4+CD25+Foxp3+ cells in 3 independent experiments with N = 6 mice total per group. C. CD4+CD25+Foxp3+ T cells in co-cultures of anti-CD3 stimulated splenocytes, IL-2 (50 ng/mL), and peritoneal cells from EPS-treated or untreated (NT) mice. D. % CD4+CD25+Foxp3+ cells in 3 independent experiments with N = 4 mice total per group.

Article Snippet: For transwell experiments, 1×10 6 splenocytes were cultured in anti-CD3-coated 24-well plates with or without transwell inserts (Corning) containing 5×10 5 peritoneal cells.

Techniques: Expressing