Journal: PLoS ONE

Article Title: IL-33 Is Produced by Mast Cells and Regulates IgE-Dependent Inflammation

doi: 10.1371/journal.pone.0011944

Figure Lengend Snippet: 1×10 6 BMMC were primed with 1 µg/ml anti-DNP IgE (SPE-7 clone) overnight and stimulated by addition of 0.5 µg/ml DNP-HSA (black bars) or vehicle control (white bars). After the indicated times, gene expression of mouse IL-33 was determined by real-time RT-PCR (A). Either anti-DNP IgE or anti-OVA IgE was used in combination with either DNP-HSA or OVA, as indicated, and IL-33 gene expression determined (B). Intracellular staining for IL-33 was determined in permeabilized BMMC 24 hours after stimulation of 0.5 µg/ml DNP-HSA (C). Western blot analysis of BMMC lysate from stimulated or unstimulated cells 24 hours after stimulation (D). Recombinant, mouse IL-33 109–266 was used as a positive control. Data represents 3 independent experiments. Primary mast cells were obtained by peritoneal lavage, identified as CD117 + /FcεRI + and intracellular staining for IL-33 performed after permeabilization (E). Data represents 2 independent experiments.

Article Snippet: Rabbit polyclonal anti-IL-33 (Axxora) (1∶500) was used as a primary detection antibody and biotinylated goat anti-rabbit IgG (Zymed) was used as a secondary antibody.

Techniques: Expressing, Quantitative RT-PCR, Staining, Western Blot, Recombinant, Positive Control

Journal: PLoS ONE

Article Title: IL-33 Is Produced by Mast Cells and Regulates IgE-Dependent Inflammation

doi: 10.1371/journal.pone.0011944

Figure Lengend Snippet: 1×10 6 BMMC were primed with 1 µg/ml anti-DNP IgE (SPE-7 clone) overnight and stimulated by addition of 0.5 µg/ml DNP-HSA or 10 ng/ml of recombinant IL-33 109–266 . After 4 hours, the gene expression of cytokines was determined by real-time RT-PCR in BMMC (A) and in MC/9 cells (B). IL-33 expression in response to ionomycin stimulation was determined using BMMC (C). The expression of IL-33 was then determined in BMMC after stimulation with 0.25 µM ionomycin or IgE/antigen in the presence or absence of 5 mM EDTA (D). * = p<0.05, ** = p<0.01 by Students t -test. Data represents the mean±SEM from 6 individual wells over two independent experiments.

Article Snippet: Rabbit polyclonal anti-IL-33 (Axxora) (1∶500) was used as a primary detection antibody and biotinylated goat anti-rabbit IgG (Zymed) was used as a secondary antibody.

Techniques: Recombinant, Expressing, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: IL-33 Is Produced by Mast Cells and Regulates IgE-Dependent Inflammation

doi: 10.1371/journal.pone.0011944

Figure Lengend Snippet: 1×10 6 BMMC (A) or MC/9 cells (B) were primed with 1 µg/ml anti-DNP IgE (SPE-7 clone) overnight and stimulated by addition of 0.5 µg/ml DNP-HSA or 10 ng/ml of recombinant IL-33 109–266 . After 4 hours, the gene expression of ST2 was determined by real-time RT-PCR. Surface expression of ST2 protein on BMMC was determined by flow cytometry after 6 hours (C). The levels of ST2 in supernatants were analyzed by ELISA after 6 hours (D). * = p<0.05, *** = p<0.005 by Students t -test. Data in A and C represents the mean±SEM from 3 to 6 individual wells over two independent experiments. Data in B is representative of results from three independent experiments.

Article Snippet: Rabbit polyclonal anti-IL-33 (Axxora) (1∶500) was used as a primary detection antibody and biotinylated goat anti-rabbit IgG (Zymed) was used as a secondary antibody.

Techniques: Recombinant, Expressing, Quantitative RT-PCR, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: IL-33 Is Produced by Mast Cells and Regulates IgE-Dependent Inflammation

doi: 10.1371/journal.pone.0011944

Figure Lengend Snippet: The tissue levels of IL-33 in the skin of wild type or mast cell deficient mice (W/Wv) were determined by ELISA (A). Additionally, frozen sections (7 µm) were stained for IL-33 immunoreactivity using biotinylated anti-IL-33 and ABC-DAB substrate and sections counterstained with hematoxylin (B). Total tissue IL-33 was determined in IgE-primed or sham skin in passive cutaneous anaphylaxis (C) and IL-33 immunoreactivity determined, as before (D). ** = p<0.01 by Students t -test. Data in panels A and C represents the mean±SEM from 5 individual mice while panels B and D are representative of results from 4 individual mice.

Article Snippet: Rabbit polyclonal anti-IL-33 (Axxora) (1∶500) was used as a primary detection antibody and biotinylated goat anti-rabbit IgG (Zymed) was used as a secondary antibody.

Techniques: Enzyme-linked Immunosorbent Assay, Staining

Journal: PLoS ONE

Article Title: IL-33 Is Produced by Mast Cells and Regulates IgE-Dependent Inflammation

doi: 10.1371/journal.pone.0011944

Figure Lengend Snippet: Induction of ear swelling during passive cutaneous anaphylaxis was determined after systemic antigen challenge in mice receiving no treatment, 10 µg anti-murine IL-33 antibody or 10 µg isotype control (A), anti-murine ST2 antibody or isotype control (B) or in ST2 −/− or ST2 −/+ control mice (C). Data represents the mean±SEM from 10–15 mice. The IL-33 protein levels in ear tissue from IgE injected or PBS injected sites were determined by ELISA during the PCA time course (D) (n = 3 mice per time point). Increases in IL-33 at IgE injected skin sites (determined as the delta between the PBS injected skin from the same individual) were determined in W/Wv, reconstituted W/Wv, ST2 −/− or relevant control strains (n = 3–9 mice per group). * = p<0.05, ** = p<0.01, *** = p<0.005 by Students t -test.

Article Snippet: Rabbit polyclonal anti-IL-33 (Axxora) (1∶500) was used as a primary detection antibody and biotinylated goat anti-rabbit IgG (Zymed) was used as a secondary antibody.

Techniques: Injection, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: IL-33 Is Produced by Mast Cells and Regulates IgE-Dependent Inflammation

doi: 10.1371/journal.pone.0011944

Figure Lengend Snippet: 1×10 6 BMMC were primed with 1 µg/ml anti-DNP IgE (SPE-7 clone) overnight and stimulated by addition of 0.5 µg/ml DNP-HSA (black bars) or vehicle control (white bars). After the indicated times, gene expression of mouse IL-33 was determined by real-time RT-PCR (A). Either anti-DNP IgE or anti-OVA IgE was used in combination with either DNP-HSA or OVA, as indicated, and IL-33 gene expression determined (B). Intracellular staining for IL-33 was determined in permeabilized BMMC 24 hours after stimulation of 0.5 µg/ml DNP-HSA (C). Western blot analysis of BMMC lysate from stimulated or unstimulated cells 24 hours after stimulation (D). Recombinant, mouse IL-33 109–266 was used as a positive control. Data represents 3 independent experiments. Primary mast cells were obtained by peritoneal lavage, identified as CD117 + /FcεRI + and intracellular staining for IL-33 performed after permeabilization (E). Data represents 2 independent experiments.

Article Snippet: Rabbit polyclonal anti-mouse IL-33 and recombinant IL-33 109–266 were obtained from Axxora.

Techniques: Expressing, Quantitative RT-PCR, Staining, Western Blot, Recombinant, Positive Control

Journal: PLoS ONE

Article Title: IL-33 Is Produced by Mast Cells and Regulates IgE-Dependent Inflammation

doi: 10.1371/journal.pone.0011944

Figure Lengend Snippet: 1×10 6 BMMC were primed with 1 µg/ml anti-DNP IgE (SPE-7 clone) overnight and stimulated by addition of 0.5 µg/ml DNP-HSA or 10 ng/ml of recombinant IL-33 109–266 . After 4 hours, the gene expression of cytokines was determined by real-time RT-PCR in BMMC (A) and in MC/9 cells (B). IL-33 expression in response to ionomycin stimulation was determined using BMMC (C). The expression of IL-33 was then determined in BMMC after stimulation with 0.25 µM ionomycin or IgE/antigen in the presence or absence of 5 mM EDTA (D). * = p<0.05, ** = p<0.01 by Students t -test. Data represents the mean±SEM from 6 individual wells over two independent experiments.

Article Snippet: Rabbit polyclonal anti-mouse IL-33 and recombinant IL-33 109–266 were obtained from Axxora.

Techniques: Recombinant, Expressing, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: IL-33 Is Produced by Mast Cells and Regulates IgE-Dependent Inflammation

doi: 10.1371/journal.pone.0011944

Figure Lengend Snippet: 1×10 6 BMMC (A) or MC/9 cells (B) were primed with 1 µg/ml anti-DNP IgE (SPE-7 clone) overnight and stimulated by addition of 0.5 µg/ml DNP-HSA or 10 ng/ml of recombinant IL-33 109–266 . After 4 hours, the gene expression of ST2 was determined by real-time RT-PCR. Surface expression of ST2 protein on BMMC was determined by flow cytometry after 6 hours (C). The levels of ST2 in supernatants were analyzed by ELISA after 6 hours (D). * = p<0.05, *** = p<0.005 by Students t -test. Data in A and C represents the mean±SEM from 3 to 6 individual wells over two independent experiments. Data in B is representative of results from three independent experiments.

Article Snippet: Rabbit polyclonal anti-mouse IL-33 and recombinant IL-33 109–266 were obtained from Axxora.

Techniques: Recombinant, Expressing, Quantitative RT-PCR, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: IL-33 Is Produced by Mast Cells and Regulates IgE-Dependent Inflammation

doi: 10.1371/journal.pone.0011944

Figure Lengend Snippet: The tissue levels of IL-33 in the skin of wild type or mast cell deficient mice (W/Wv) were determined by ELISA (A). Additionally, frozen sections (7 µm) were stained for IL-33 immunoreactivity using biotinylated anti-IL-33 and ABC-DAB substrate and sections counterstained with hematoxylin (B). Total tissue IL-33 was determined in IgE-primed or sham skin in passive cutaneous anaphylaxis (C) and IL-33 immunoreactivity determined, as before (D). ** = p<0.01 by Students t -test. Data in panels A and C represents the mean±SEM from 5 individual mice while panels B and D are representative of results from 4 individual mice.

Article Snippet: Rabbit polyclonal anti-mouse IL-33 and recombinant IL-33 109–266 were obtained from Axxora.

Techniques: Enzyme-linked Immunosorbent Assay, Staining

Journal: PLoS ONE

Article Title: IL-33 Is Produced by Mast Cells and Regulates IgE-Dependent Inflammation

doi: 10.1371/journal.pone.0011944

Figure Lengend Snippet: Induction of ear swelling during passive cutaneous anaphylaxis was determined after systemic antigen challenge in mice receiving no treatment, 10 µg anti-murine IL-33 antibody or 10 µg isotype control (A), anti-murine ST2 antibody or isotype control (B) or in ST2 −/− or ST2 −/+ control mice (C). Data represents the mean±SEM from 10–15 mice. The IL-33 protein levels in ear tissue from IgE injected or PBS injected sites were determined by ELISA during the PCA time course (D) (n = 3 mice per time point). Increases in IL-33 at IgE injected skin sites (determined as the delta between the PBS injected skin from the same individual) were determined in W/Wv, reconstituted W/Wv, ST2 −/− or relevant control strains (n = 3–9 mice per group). * = p<0.05, ** = p<0.01, *** = p<0.005 by Students t -test.

Article Snippet: Rabbit polyclonal anti-mouse IL-33 and recombinant IL-33 109–266 were obtained from Axxora.

Techniques: Injection, Enzyme-linked Immunosorbent Assay