Journal:

Article Title: Expanded B cell population blocks regulatory T cells and exacerbates ileitis in a murine model of Crohn disease

doi: 10.1172/JCI200420855

Figure Lengend Snippet: SAMP1/YitFc MLN αEβ7+CD4+ T cells possess a regulatory phenotype. (A) Comparison of the percentage of MLN cells, determined by flow cytometry, that express high levels of CD69 (n = 9 mice), CD25 (n = 9), L-selectin (n = 14), and CD45RB (n = 8) within the αEβ7+CD4+ and αEβ7–CD4+ T cell subsets. (B) Levels of secreted TNF-α, IL-2, and IL-10, measured by cytometric bead array in triplicate, from 48-hour cultures of SAMP1/YitFc MLN unfractionated CD4+ T cells (Total CD4+), αE+CD4+ T cells, or αE–CD4+ T cells (105 cells/well), stimulated with immobilized anti-CD3 and soluble anti-CD28. Values represent the averages of three independent experiments for each group. *Significantly different (P < 0.05) compared to αEβ7–CD4+ T cell population.

Article Snippet: MLN immunoglobulin concentrations were determined with the mouse immunoglobulin isotyping cytometric bead array (BD Biosciences — Pharmingen).

Techniques: Flow Cytometry

Journal:

Article Title: Expanded B cell population blocks regulatory T cells and exacerbates ileitis in a murine model of Crohn disease

doi: 10.1172/JCI200420855

Figure Lengend Snippet: Soluble IgA production increased in SAMP1/YitFc versus AKR mice. (A) Representative dot plots of κ (1:100 dilution, FL2) and λ (1:1, FL1) light chain antibody isotype levels within MLN supernatants from AKR mice (n = 2) and SAMP1/YitFc mice (n = 2), measured with a cytometric bead array containing isotype-specific beads with preset FL3 intensities. MLNs were crushed, resuspended in 5 ml, and centrifuged to remove the cell pellet from the tested supernatants. An increase of at least twofold in IgG1 κ, IgA κ, IgM κ, IgA λ, and IgM λ was seen in SAMP1/YitFc versus AKR MLNs, as measured by mean fluorescence intensity of the isotype-specific beads. (B) ELISA detecting IgA antibody concentrations (mean ± SEM) in serum samples collected via cardiac puncture from SAMP1/YitFc mice (n = 12) and wild-type AKR mice (n = 13). (C) Concentrations of IgA (n = 4), IgM (n = 2), and IgG2a (n = 2), measured in triplicate by ELISA, from 3-, 7-, and 11-day anti-CD3–stimulated cocultures of SAMP1/YitFc CD4+ T cells and B cells versus AKR CD4+ T cells and B cells (105 T cells/well and 105 B cells/well). *Significantly greater than AKR concentrations (P < 0.05).

Article Snippet: MLN immunoglobulin concentrations were determined with the mouse immunoglobulin isotyping cytometric bead array (BD Biosciences — Pharmingen).

Techniques: Fluorescence, Enzyme-linked Immunosorbent Assay