Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: An IL-1 cytokine member, IL-33, induces human basophil activation via its ST2 receptor.

doi: 10.4049/jimmunol.181.9.5981

Figure Lengend Snippet: FIGURE 1. Real-time quantitative PCR and flow cytometric analysis for ST2. A, cDNAs from highly purified basophils (n 7), eosinophils (n 7), and neutrophils (n 7) were tested. The data are expressed as the following ratio: copy number of ST2 gene/copy number of -actin gene 10,000. , p 0.05 and , p 0.001 vs the ratio of basophils. Data for the positive-control ST2-expressing cells, i.e., cultured mast cells, are reported in our recent article (27) and included in this figure (n 4). B, Highly purified basophils were incubated with and without IL-33 at 100 ng/ml for 4 h before RNA extraction (n 5). The calculated ratios for the same donor are connected with a solid line in the graph. C, Highly purified basophils were stained for surface-expressed ST2. Cells stained with control Ab are shown as a shaded area. D, Purified basophils were cultured with 300 pM IL-3 for 18 h and then stained for surface-expressed ST2. The cells incubated in medium alone are shown as a thin line, and the cells cultured with IL-3 are shown as a thick line. The cells stained with control Ab are shown as a shaded area. E, Intracellular ST2 staining of freshly isolated basophils. The cells stained with control Ab are shown as a shaded area. All the flow cytometry data are representative of three separate experiments using cells from different donors and showing similar results.

Article Snippet: The following Abs were purchased as indicated: mouse anti-IL-4 mAb (IgG1, clone 8D4–8) and biotin-conjugated rat anti-IL-4 mAb (IgG1, clone MP4–25D2) (eBioscience); FITC-conjugated goat anti-human IgE Ab (Biosource International); mouse neutralizing anti-ST2 mAb (IgG1, clone 97203) (R&D Systems); rabbit anti-IL-33 pAb (Adipogen); mouse anti-ST2 mAb (IgG1, clone HB12) (MBL); control mouse IgG1 (MOPC21) and mouse IgG2a (UPC10) (Sigma-Aldrich); mouse IgG2b mAb (MOPC 195) (Cappel); and FITC-conjugated goat anti-mouse IgG (Jackson ImmunoResearch).

Techniques: Real-time Polymerase Chain Reaction, Positive Control, Expressing, Cell Culture, Incubation, RNA Extraction, Staining, Control, Isolation, Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: An IL-1 cytokine member, IL-33, induces human basophil activation via its ST2 receptor.

doi: 10.4049/jimmunol.181.9.5981

Figure Lengend Snippet: FIGURE 4. IL-33 enhances basophil CD11b expression through their surface ST2 receptor. A, Percoll-separated basophils were incubated with medium alone (thin line) or IL-33 at 10 ng/ml (thick line) for 30 min. Then the surface CD11b expression level was assessed by flow cytometry. Data are representative of three separate experiments yielding similar results. B, Percoll-separated basophils were incubated with and without cytokines for 30 min, and then the levels of surface CD11b expression were analyzed. The data are expressed as the percentages of MESF values of basophils treated without any stimulus (nil). Error bars represent the SEM (n 3). , p 0.05; , p 0.01 vs nil. C, Basophils were incubated with the indicated concentrations of IL-33 for either 30 min () or 18 h (f). Then the surface CD11b expression level was assessed. Error bars represent the SEM (n 5). , p 0.001 vs nil. D, Basophils were incubated with IL-33 plus either IL-3 (u) or CRA-1 mAb (f) for 30 min, and then the expression level of CD11b was quantified. Error bars represent the SEM (n 3–6). , p 0.01; , p 0.001 vs corresponding values of cells cultured without IL-33. E, Basophils were preincubated with medium alone (u), control IgG (), or neutralizing anti-ST2 Ab (f) at 10 g/ml for 60 min and then treated with IL-33 at indicated concentrations for 30 min. Error bars represent the SEM (n 3). , p 0.05; , p 0.001 vs nil.

Article Snippet: The following Abs were purchased as indicated: mouse anti-IL-4 mAb (IgG1, clone 8D4–8) and biotin-conjugated rat anti-IL-4 mAb (IgG1, clone MP4–25D2) (eBioscience); FITC-conjugated goat anti-human IgE Ab (Biosource International); mouse neutralizing anti-ST2 mAb (IgG1, clone 97203) (R&D Systems); rabbit anti-IL-33 pAb (Adipogen); mouse anti-ST2 mAb (IgG1, clone HB12) (MBL); control mouse IgG1 (MOPC21) and mouse IgG2a (UPC10) (Sigma-Aldrich); mouse IgG2b mAb (MOPC 195) (Cappel); and FITC-conjugated goat anti-mouse IgG (Jackson ImmunoResearch).

Techniques: Expressing, Incubation, Cytometry, Cell Culture, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: An IL-1 cytokine member, IL-33, induces human basophil activation via its ST2 receptor.

doi: 10.4049/jimmunol.181.9.5981

Figure Lengend Snippet: FIGURE 5. IL-33 enhances human basophil migration toward eotaxin. A, Two 104 Percoll-separated basophils were added to the upper cham- ber. IL-33 at 10 or 100 ng/ml or eotaxin (Eot) at 10 nM was added to the lower chamber. Cells mixed with IL-33 at 10 ng/ml were also tested for migration toward eotaxin. The percentage of migrated cells was calculated by subtracting the spontaneous migration (9.6 0.4% for medium only). Error bars represent the SEM (n 5). , p 0.01 vs spontaneous mi- gration in medium alone. B, Percoll-separated basophils were mixed with the indicated concentrations of IL-33 and then tested for migration toward eotaxin at 10 nM. The percentage of migrated cells was calculated by subtracting the spontaneous migration (16.8 0.5% for medium only). Error bars represent the SEM (n 3). , p 0.01 vs basophil migration toward eotaxin in the absence of IL-33. C, Both Percoll-separated () and MACS-separated (f) basophils were used for the migration assay. Baso- phil preparations with and without IL-33 at 100 ng/ml were placed in the upper chamber, and eotaxin at 50 nM was added to the lower chamber. The percentage of migrated cells was calculated by subtracting the spontaneous migration (16.8 0.8% for Percoll-separated and 12.3 1.2% for MACS- separated preparations). Error bars represent the SEM (n 3). , p 0.01 vs migration of the corresponding basophils without IL-33. D, Effect of neutralizing Ab for ST2 on IL-33 enhancement of basophil migration. Per- coll-separated basophils were mixed without Ab () or with control IgG at 20 g/ml (u) or anti-ST2 Ab at 20 g/ml (f). IL-33 at 10 ng/ml was then added to the cells; eotaxin at 10 nM was added to the lower chamber. Data shown are mean values of an experiment performed in duplicate. Another experiment using basophils from a different donor yielded similar results. E, Effect of IL-33 on CCR3 expression by human basophils. Highly puri- fied basophils were incubated with (bold line) and without (thin line) IL-33 at 10 ng/ml for 1 h at 37°C. The dotted line indicates basophils that were incubated with IL-3 at 300 pM. Cells stained with control Ab are shown as a shaded area. Data are representative of two separate experiments using cells from different donors and showing similar results.

Article Snippet: The following Abs were purchased as indicated: mouse anti-IL-4 mAb (IgG1, clone 8D4–8) and biotin-conjugated rat anti-IL-4 mAb (IgG1, clone MP4–25D2) (eBioscience); FITC-conjugated goat anti-human IgE Ab (Biosource International); mouse neutralizing anti-ST2 mAb (IgG1, clone 97203) (R&D Systems); rabbit anti-IL-33 pAb (Adipogen); mouse anti-ST2 mAb (IgG1, clone HB12) (MBL); control mouse IgG1 (MOPC21) and mouse IgG2a (UPC10) (Sigma-Aldrich); mouse IgG2b mAb (MOPC 195) (Cappel); and FITC-conjugated goat anti-mouse IgG (Jackson ImmunoResearch).

Techniques: Migration, Control, Expressing, Incubation, Staining

Journal: International Journal of Molecular Sciences

Article Title: Regulatory T Cells Exhibit Interleukin-33-Dependent Migratory Behavior during Skin Barrier Disruption

doi: 10.3390/ijms22147443

Figure Lengend Snippet: Effects of IL-33 on Treg migration. ( a ) mRNA expression by ST2 + Foxp3 + Treg cells detected by in situ hybridization; n = 6–8. *** p < 0.001 compared with naïve or vehicle-treated mice by the Kruskal–Wallis with Dunn’s multiple-comparison test. Scale bars, 100 μm. ( b ) Transwell migration assay of Tregs was performed. The number of cells that migrated into the lower chamber after a 2-h incubation at 37 °C is shown; n = 2–8 chambers in each group. Numbers represent the mean ± SEM of five independent experiments. * p < 0.05 and ** p < 0.01 compared with vehicle by the Kruskal–Wallis with Dunn’s multiple-comparison test.

Article Snippet: To detection of ST2 + and Foxp3 + cells, spleen cells were suspended in HBSS containing 2% FCS passed through a 70-μm pore size nylon mesh, and stained with FITC-conjugated ST2 mAb (DJ8; MD Biosciences; Oakdale, MN, USA) or Alexa Fluor ® 488-conjugated Foxp3.

Techniques: Migration, Expressing, In Situ Hybridization, Comparison, Transwell Migration Assay, Incubation

Journal: Cell

Article Title: Homeostatic Control of Sebaceous Glands by Innate Lymphoid Cells Regulates Commensal Bacteria Equilibrium

doi: 10.1016/j.cell.2018.12.031

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: PE T1/ST2 (IL-33 R) Monoclonal Antibody , MD Biosciences , Cat# 101001PE.

Techniques: Purification, Virus, Isolation, Recombinant, Staining, Transfection, Electron Microscopy, dsDNA Assay, Software