Journal: Journal of immunology (Baltimore, Md. : 1950)
Article Title: CD40 Ligand and Appropriate Cytokines Induce Switching to IgG, IgA, and IgE and Coordinated Germinal Center and Plasmacytoid Phenotypic Differentiation in a Human Monoclonal IgM+IgD+ B Cell Line
Figure Lengend Snippet: Fluorescence flow cytometric analysis of the expression of sIgM, sIgG, sIgA, and sIgE by CL-01 cells and analysis of secreted IgM, IgG, IgA, and IgE. Dot plots depict the expression of sIgM, sIgD, sIgG, and sIgA by CL-01 cells (day 0) and by CL-01 cells cocultured with CD40L-293 cells and IL-4 (100 U/ml) for 5 or 8 days. Numbers inside quadrants are percentages of the total cells. Results are representative of three independent experiments. Histograms show the concentration of IgM (■), IgG ( ), IgA (▨), and IgE (□) accumulated in the fluids of CL-01 cells incubated for 8 days with or without IL-4, and cultured alone, with CD8-293 cells, with CD40L-293 cells, and with CD40L-293 cells in the presence of neutralizing anti-IL-10 and/or anti-TGF- β Abs (the concentrations of IgM and IgE were from cultures with both anti-IL-10 and anti-TGF- β Abs, whereas the concentrations of IgG and IgA were from cultures with anti-IL-10 and anti-TGF- β Abs, respectively). Values are mean ± SD of four determinations from three independent experiments.
Article Snippet: Following rinsing with TBST buffer (10 mM Tris-HCl (pH 8), 150 mM NaCl, 0.02% Tween-20, and 0.02% NaN3 ) and incubation with TBST containing 3% BSA, cells were stained with 4′,6-diamidine-2′-phenylindole dihydrochloride (Boehringer Mannheim Corp., Indianapolis, IN), rhodamine-conjugated goat Ab to human IgG (ICN Pharmaceuticals, Inc., Costa Mesa, CA), and FITC-conjugated mouse mAb to human IgA (Sigma Chemical Co., St. Louis, MO).
Techniques: Fluorescence, Flow Cytometry, Expressing, Concentration Assay, Incubation, Cell Culture