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Blood, Vol. 113, Issue 17, 3969-3977, April 23, 2009
BCR-mediated uptake of antigen linked to TLR9 ligand stimulates B-cell proliferation and antigen-specific plasma cell formation
Blood Eckl-Dorna and Batista
113: 3969
Supplemental materials for: Eckl-Dorna and Batista
Files in this Data Supplement:
- Figure S1. Stimulation with HEL-CpG particulates gives rise to sustained p38 phosphorylation and is dependent on a functional TLR9 in vitro (JPG, 104 KB)
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(A) Biotinylated Ags (left panel) OVA and (right panel) CγG were immobilised on streptavidin-coated particles and were visualized by flow cytometry following binding of anti-OVA followed by anti-Mouse IgG-AlexaFluor-488 or anti-CγG FITC in the presence (filled grey) or absence (black line) of CpG. (B) Binding of biotinylated HEL to streptavidin-coated microspheres in the presence (grey filled) or absence (black line) of CpG was detected by: (upper panel) Western blotting using a polyclonal anti-HEL and (lower panel) flow cytometry via binding of anti-HEL F10 AlexaFluor-488. (C) MD4 B cells (upper panel) and bone marrow derived DCs (lower panel) were either not stimulated (filled grey) or stimulated with 0.2 µl particulates that were coated with HEL alone, CpG alone or HEL-CpG (solid line). Flow cytometry was used to assess binding of particulates. Gates indicate percentage of live cells binding to the particulates. (D) CFSE-labelled (left panel) MD4 MyD88−∕− or (right panel) MD4 TLR9−∕− (grey filled) B cells were stimulated with 1 µl particulate HEL alone (grey filled) or particulate HEL-CpG (black line). Three days after stimulation (upper panels) CFSE dilution and (lower panels) CD138 upregulation were assessed by flow cytometry. (E) MD4 B cells were stimulated with 1 µl particulate HEL alone (open bars), particulate CpG alone (grey bars) or particulate HEL-CpG (filled bars). Samples were taken at the indicated times after stimulation, and equal amounts of whole cell-lysates were subjected to SDS-PAGE. (Upper panel) Subsequently Western blotting was performed using specific antibodies to phosphorylated p38 (pp38), total p38 and actin (loading control) for detection. (Lower panel) The density of the bands was quantified by densitometry, corrected for background, normalized to the density of the actin band in the same sample, and then made relative to the unstimulated zero time point for each condition.
- Figure S2. Stimulation of B-cell proliferation and differentiation by Ag-CpG particulates is dependent on Ag avidity in vitro (JPG, 50.4 KB)
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(A, B) MD4 B cells were stimulated with 1 µl particulate CpG coated together with either (left panels) HEL or (right panels) HELK at (upper panels and filled bars) high or (lower panels and open bars) low density for 72h. (A) CFSE dilution of stimulated (black line) and unstimulated cells (filled grey) B cells was assessed by flow cytometry. (B) (Left panel) IL-6 and (right panel) IgMa secretion were assessed by ELISA. (C) Streptavidin-coated microspheres were coated with different densities of biotinylated anti-HEL F10 (grey scale). Binding of F10 was visualized was visualized by flow cytometry using anti-mouse IgG AlexaFluor-488.
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