Journal: Immunology

Article Title: Expression and regulation of interleukin-33 in human monocytes

doi: 10.1111/j.1365-2567.2009.03221.x

Figure Lengend Snippet: Interleukin-33 (IL-33) protein is closely associated with the nucleus in THP-1 monocytes undergoing apoptosis. Immunocytochemistry was performed on unstimulated THP-1 monocytes (control) and on cells stimulated with 100 ng/ml Escherichia coli (Ec) or Porphyromonas gingivalis (Pg) lipopolysaccharide (LPS) for 9 hr followed by exposure to UVB-irradiation to induce apoptosis. The IL-33 was located using an IL-33 antibody and secondary antibody conjugated to fluorescein isothiocyanate (FITC; green). The nuclei were stained with 4′-6′,diamidino-2-phenylindole (DAPI; blue). The upper panels (a) show IL-33 expression alone (green FITC) and the lower panels (b) show IL-33 (green FITC) and nuclei (blue DAPI) in combination. Arrows indicate examples of apoptotic cells. The results are representative of three independent stimulation experiments and have been verified by an independent observer. Scale as depicted on the images.

Article Snippet: The cells were then probed with 5 μg/ml of a mouse monoclonal antibody to IL-33 (Nessy-1, Axxora, Nottingham, UK) for 60 min at room temperature.

Techniques: Immunocytochemistry, Irradiation, Staining, Expressing

Journal: Immunology

Article Title: Expression and regulation of interleukin-33 in human monocytes

doi: 10.1111/j.1365-2567.2009.03221.x

Figure Lengend Snippet: Interleukin-33 (IL-33) messenger RNA (mRNA) expression is up-regulated by monocytes in response to lipopolysaccharide (LPS) but not tumour necrosis factor-α (TNF-α) and IL-1β. Quantitative real-time polymerase chain reaction analysis was used to determine IL-33 expression by THP-1 monocytes (a) and primary human monocytes (b) in response to stimulation with 100 ng/ml Escherichia coli or Porphyromonas gingivalis LPS. IL-33 mRNA expression by THP-1 monocytes stimulated with 100 ng/ml TNF-α and 100 pg/ml IL-1β was also measured (c). Levels of mRNA were normalized against RNA polymerase II mRNA levels and fold changes (‘IL-33 expression’) were calculated relative to unstimulated cells at each individual time-point using the ΔΔCt method.20 The data represent the mean values (± SD) from three independent experiments. Significant differences we determined using Student’s t-test on the ΔCt values.20*P < 0·05 relative to unstimulated cells.

Article Snippet: The cells were then probed with 5 μg/ml of a mouse monoclonal antibody to IL-33 (Nessy-1, Axxora, Nottingham, UK) for 60 min at room temperature.

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: Immunology

Article Title: Expression and regulation of interleukin-33 in human monocytes

doi: 10.1111/j.1365-2567.2009.03221.x

Figure Lengend Snippet: Interleukin-33 (IL-33) is not secreted by viable THP-1 monocytes but is released from necrotic cells. THP-1 monocytes were either stimulated with 100 ng/ml Escherichia coli or Porphyromonas gingivalis lipopolysaccharide (LPS) for 9 hr (live control), or stimulated with 100 ng/ml E. coli or P. gingivalis LPS for 3–48 hr followed by induction of necrosis by five rounds of freezing (− 70°) and thawing (38°). Stimulation of the cells in these experiments was confirmed by detection of tumour necrosis factor-α in the isolated supernatants using enzyme-linked immunosorbent assay (not shown). ND = not detected. The data represent the mean values (± SD) from three independent experiments.

Article Snippet: The cells were then probed with 5 μg/ml of a mouse monoclonal antibody to IL-33 (Nessy-1, Axxora, Nottingham, UK) for 60 min at room temperature.

Techniques: Isolation, Enzyme-linked Immunosorbent Assay

Journal: Immunology

Article Title: Expression and regulation of interleukin-33 in human monocytes

doi: 10.1111/j.1365-2567.2009.03221.x

Figure Lengend Snippet: Interleukin-33 (IL-33) protein is expressed in the cytoplasm of lipopolysaccharide (LPS) -stimulated THP-1 monocytes (a) and primary monocytes (b). Immunocytochemistry was performed on monocytes (control) and cells stimulated with 100 ng/ml Escherichia coli (Ec) or Porphyromonas gingivalis (Pg) LPS for 9 hr. The IL-33 was located using an IL-33 antibody and secondary antibody conjugated to fluorescein isothiocyanate (FITC; green) or tetramethyl rhodamine iso-thiocyanate (TRITC; red) and the nuclei were stained with 4′-6′,diamidino-2-phenylindole (DAPI; blue). A series of Z-stack images were taken to confirm the cellular location of IL-33 and the image depicted is representative of findings throughout the Z stack. The upper panels (a) show IL-33 expression alone (green FITC) and the lower panels (b) show IL-33 (red TRITC) and nuclei (blue DAPI) in combination. The results are representative of three independent stimulation experiments and have been verified by an independent observer. Scale as depicted on images.

Article Snippet: The cells were then probed with 5 μg/ml of a mouse monoclonal antibody to IL-33 (Nessy-1, Axxora, Nottingham, UK) for 60 min at room temperature.

Techniques: Immunocytochemistry, Staining, Expressing