Il 33

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom, Germany

IL-33 is a recombinant human interleukin-33 protein. Interleukin-33 is a cytokine that plays a role in immune and inflammatory responses.

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117 protocols using il 33

Analyzing NF-κB Signaling in Mast Cells

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HEK293T cells were washed with ice cold PBS before lysis. HMC-1.1 and HMC-1.2 cells (10⁶ cells/ml) were treated with vehicle (DMSO) or inhibitors (60 min) (Merck Millipore). HMC-1.1 cells were then left unstimulated or were stimulated with IL-33 (peprotech). Ba/F3 cells, NF-κB-EGFP-MC/9 cells or BMMCs (10⁶ cells/ml) were IL-3-starved (1 h). Afterwards, Ba/F3 cells were treated with DMSO or the IKK-inhibitor VII (60 min). MC/9 cells and BMMCs were DMSO- or inhibitor-treated (30 min) and were single (SCF or IL-33) or co-stimulated (SCF and IL-33) (peprotech). Cells were lysed (20 mM HEPES, pH7.5; 10 mM EDTA; 40 mM β-glycerophosphate; 2,5 mM MgCl₂; 2 mM orthovanadate; 1 mM dithiothreitol; 20 μg/ml aprotinin; 20 μg/ml leupeptin supplemented with 1% Triton) and protein concentration was determined by using the BCA-kit (Pierce). Then protein samples were boiled in 6 × Laemmli buffer.

Drube S., Weber F., Göpfert C., Loschinski R., Rothe M., Boelke F., Diamanti M.A., Löhn T., Ruth J., Schütz D., Häfner N., Greten F.R., Stumm R., Hartmann K., Krämer O.H., Dudeck A, & Kamradt T. (2015). TAK1 and IKK2, novel mediators of SCF-induced signaling and potential targets for c-Kit-driven diseases. Oncotarget, 6(30), 28833-28850.

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Multiplexed ELISA for Cytokine Quantification

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96-well plates were coated with the appropriate capture antibodies: IL-8 (M801; Thermo Fisher Scientific, USA) and IL-10 (BioLegend, USA) overnight at 4°C and IL-33, VEGF, TGF-β1 (all R&D, USA) overnight at room temperature. On the next day, plates were washed with wash buffer (PBS-Tween 0.05%), incubated with blocking buffer (IL-8: 4% BSA in PBS-Tween 0.05%) or reagent diluent (IL-33, VEGF: 1% BSA in PBS) or block buffer (TGF-β1: 5% Tween-PBS) or assay diluent (IL-10) for 1 hour at room temperature. Standards and samples were applied to plates and incubated for either 1 hour (IL-8) or 2 hours (IL-33, VEGF, TGF-β1, and IL-10). The activation of latent TGF-β1 in supernatants was assessed by adding 1 N HCl for 10 minutes and stopped with 1.2 N NaOH/0.5 M HEPES. Next, the respective detection antibodies were incubated for either 1 hour (IL-8 and IL-10) or 2 hours (IL-33, VEGF, and TGF-β1) and subsequently incubated with a streptavidin-HRP for 20-30 minutes. TMB substrate solution (Thermo Fisher Scientific) was added and incubated for 20 minutes (IL-8, IL-33, VEGF and TGF-β1) or 30 minutes (IL-10) in dark at room temperature. After adding 0.18 M H₂SO₄ (IL-8) and 2 N H₂SO₄ (IL-33, VEGF, TGF-β1, and IL-10) to stop the reaction, the optical densities at 450 nm were measured using Multiskan™ FC Microplate Photometer (Thermo Fisher Scientific).

Nikolić N., Kienzl P., Tajpara P., Vierhapper M., Matiasek J, & Elbe-Bürger A. (2019). The Antiseptic Octenidine Inhibits Langerhans Cell Activation and Modulates Cytokine Expression upon Superficial Wounding with Tape Stripping. Journal of Immunology Research, 2019, 5143635.

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Eosinophil-Macrophage Interaction Assay

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Eosinophils were isolated as described previously (25 (link)) from NJ.1638 or IL-13^−/−/NJ.1638 (23 (link), 24 (link)) mice. IL-33 (Peprotech) was added to activate the eosinophils to 50ng/mL (IL-33 was omitted from the resting eosinophil experimental group). Cells were incubated at 37°C, 5%CO₂ and 24 hours later were washed three times to remove added cytokines. Macrophages were isolated from C57BL/6J or MMP-12^−/− mice as described by Lasbury et al (26 (link)). Cells were plated at 150,000 cells per well in 24 well culture plates (Corning, Corning, NY) and cultured for 48 hours with the addition of either eosinophils (750,000 cells) or media alone in 500μl total volume. To assess contact dependency the above was performed in transwell plates (0.4μm pore) (Corning).

Doyle A.D., Mukherjee M., LeSuer W.E., Bittner T.B., Pasha S.M., Frere J.J., Neely J.L., Kloeber J.A., Shim K.P., Ochkur S.I., Ho T., Svenningsen S., Wright B.L., Rank M.A., Lee J.J., Nair P, & Jacobsen E.A. (2019). Eosinophil-derived IL-13 Promotes Emphysema. The European respiratory journal, 53(5), 1801291.

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Differentiation of ILC2 Progenitors

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The ILC2 differentiation potential of hematopoietic progenitors was performed as previously reported (Wong et al., 2012 (link)). In brief, freshly sorted CLPs from the Bcl11b^flox/floxRosa26^{CreERT2/CreERT2} and control Bcl11b^+/+ Rosa26^{CreERT2/CreERT2} mice that were treated with Tam 4 d earlier (4.0 mg Tam by intraperitoneal injection over three consecutive days) were cultured on OP9-DL1 monolayers in the presence of 10 ng/ml IL-7 (PeproTech) and 10 ng/ml IL-33 (PeproTech) for 22 d.
For the short-term fate assay, purified Bcl11b⁻ChILPs and Bcl11b⁺ChILPs were cultured on OP9 monolayers in the presence of 25 ng/ml IL-7 (PeproTech) and 25 ng/ml Stem Cell Factor (SCF; PeproTech) for 6 d as previously described (Constantinides et al., 2014 (link)).
For deleting Bcl11b in ILC2s, sorted ILC2Ps from the BM of Bcl11b^floxfloxRosa26^{CreERT2/CreERT2} and control mice were cultured on OP9-DL1 monolayers in the presence of 20 ng/ml IL-7, 20 ng/ml SCF, and 10 ng/ml IL-2 or 20 ng/ml IL-7 plus 20 ng/ml IL-25 or plus 20 ng/ml IL-33. After 3–5 d, Tam was added in the medium to induce Bcl11b deletion in vitro. Cells were collected and analyzed 10–14 d after Tam treatment.

Yu Y., Wang C., Clare S., Wang J., Lee S.C., Brandt C., Burke S., Lu L., He D., Jenkins N.A., Copeland N.G., Dougan G, & Liu P. (2015). The transcription factor Bcl11b is specifically expressed in group 2 innate lymphoid cells and is essential for their development. The Journal of Experimental Medicine, 212(6), 865-874.

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Isolation and Activation of Murine BMDMs

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Murine bone marrow-derived macrophages (BMDM) were isolated and characterized as previously described(22 (link)). Briefly, bone marrow was harvested from 6- to 8-week-old B6 mice, B6 st2^−/− mice, or Balb/c st2^−/− mice. Harvested cells from the bone marrow were washed and plated at 2 × 10⁶ cells/mL and were allowed to differentiate into macrophages for 7 days in the presence of macrophage colony-stimulating factor (MCSF) with complete medium changes every 48 h. Macrophages were then activated for 24 h with one of the following: 1) 20 ng/mL Interferon-γ (IFNγ) and 100 ng/mL lipopolysaccharide (LPS) (Affymetrix eBioscience, Santa Clara, CA; Sigma Aldrich) to promote an M_IFNγ+LPS phenotype (M1-like); 2) 20 ng/mL interleukin (IL)-4 (Invitrogen) to promote an M_IL-4 phenotype (M2-like); 3) 20 ng/ml IL-33 (Peprotech), or 4) 25 µg/mL of wt mouse MBV, IL-33^−/− mouse MBV, or porcine SIS-MBV. After the incubation period at 37°C, cells were washed with sterile PBS and lysates prepared using RIPA buffer for immunoblot analysis, or cells fixed with 2% paraformaldehyde (PFA) for immunolabeling.

Hussey G.S., Dziki J.L., Lee Y.C., Bartolacci J.G., Behun M., Turnquist H.R, & Badylak S.F. (2019). Matrix bound nanovesicle-associated IL-33 activates a pro-remodeling macrophage phenotype via a non-canonical, ST2-independent pathway. Journal of immunology and regenerative medicine, 3, 26-35.

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Modulation of ILC2 Function by IL-33 and G-CSF

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Mice were administered 1 μg recombinant IL-33 (ThermoFisher Scientific) for 1 week as detailed above in “recombinant IL-33 and G-CSF administration”. At 24 hours after the final IL-33 dose, lung tissue was extracted and single cell suspensions were obtained as described above in “Cell recovery and isolation”. ILC2s were sorted as described in “FACS of T cells and ILC2s”. Post sort, the isolated ILC2s were immediately plated in medium containing DMEM + 10% FCS, IL-7 (5 ng/ml; PeproTech) containing IL-33 (10 ng/ml; PeproTech) or lacking IL-33. Respective wells were subsequently supplemented with either media, 10 ng/ml G-CSF or 100 ng/ml G-CSF for 72 hours. After 72 hours, the supernatant was collected for the measurement of IL-5 and IL-13 by ELISA and the cells were lysed in 350 μl RLT buffer (Qiagen) for RNA analysis. The lysed cells were assessed for mRNA expression of Il5, Il13 and Gata3.

Patel D.F., Peiró T., Bruno N., Vuononvirta J., Akthar S., Puttur F., Pyle C.J., Suveizdytė K., Walker S.A., Singanayagam A., Carlin L.M., Gregory L.G., Lloyd C.M, & Snelgrove R.J. (2019). Neutrophils restrain allergic airway inflammation by limiting ILC2 function and monocyte-dendritic cell antigen presentation. Science immunology, 4(41), eaax7006.

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Activation of Mucosal Mast Cells by IL-33

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BMMCs were plated at a density of 5×10⁴ cells per ml in a 96 U-bottom well plate in a total of 200 µl complete RPMI per well and incubated overnight, with 50 ng/ml IgE anti-dinitrophenyl (DNP) (clone SPE-7, Sigma-Aldrich) in the presence of the following: medium, 100 ng/ml IL-33 (Peprotech), IL-33 and 5 µg/ml of anti-ST2 mAb DJ8 or isotype control mAb. The next day 50 ng/ml DNP conjugated human serum albumin (DNP-HSA, Sigma-Aldrich) or medium were added and 10 min later the cells were incubated for 15 min on ice with TruStain FcX, then stained for 20 min with eF506 viability dye from eBioscience, BV421 anti-IgE (R35–72) from BD Horizon and APC-Cy7 anti-LAMP-1 (1D4B) from Biolegend and washed. Live cells were pre-gated and analyzed as described above.

Galand C., Leyva-Castillo J.M., Yoon J., Han A., Lee M.S., McKenzie A., Stassen M., Oyoshi M.K., Finkelman F.D, & Geha R.S. (2016). IL-33 promotes food anaphylaxis in epicutaneously-sensitized mice by targeting mast cells. The Journal of allergy and clinical immunology, 138(5), 1356-1366.

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Allergic Inflammation Modulation by IL-33

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Mice were sensitized and challenged using a previously described protocol known to induce mast cell-dependent allergic inflammation^{22 (link)}. To study the effects of IL-33, the protocol was modified by introducing intranasal IL-33 instillations to sensitized mice before each antigen challenge. Mice were sensitized via i.p. injections with 10 µg/dose OVA (grade V; Sigma-Aldrich) in 100 µL saline or sham-sensitized with the equivalent volume of saline alone on days 0, 2, 4, 6, 8, 10 and 12 (Fig. 1a). The mice were then challenged with 200 µg/dose OVA in 20 µL PBS or PBS alone via i.n. instillation on days 39, 42 and 45. IL-33 (eBioscience, San Diego, CA, USA) (0.2 µg of IL-33 in 20 µL PBS) or PBS alone were given, before each OVA-challenge, via i.n. instillation on days 37, 38, 41 and 44. Lung mechanics measurements and tissue collection were performed 24 hours following the final OVA challenge.

Sjöberg L.C., Nilsson A.Z., Lei Y., Gregory J.A., Adner M, & Nilsson G.P. (2017). Interleukin 33 exacerbates antigen driven airway hyperresponsiveness, inflammation and remodeling in a mouse model of asthma. Scientific Reports, 7, 4219.

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Cytokine-Induced Immune Cell Activation

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The cells were stimulated with cytokines followed by detection of adhesion molecule expression. Fifty µl whole blood was incubated with IL‐3 (10 ng/ml, Sigma‐Aldrich), IL‐8 (100 ng/ml, R&D Systems) or IL‐33 (10 ng/ml, Thermo Fisher), diluted in RPMI, at 37° for 30 min, with RPMI as control. The stimulation was stopped by placing the cells on ice followed by incubation with the recommended concentration of antibodies for the basophil or the neutrophil panels for 30 min away from light. The RBCs were then lysed with 2 ml cold lysis buffer, and the samples were centrifuged for 5 min at 300× g at 4°, before washing with cold PBS and resuspended in 300 μl of cold PBS and subsequently analysed. The surface marker expression of CD62L, CD11b and CD49d on basophils and neutrophils was analysed by flow cytometry (Navios, Beckman Coulter).

Kalm F., Mansouri L., Russom A., Lundahl J, & Nopp A. (2020). Adhesion molecule cross‐linking and cytokine exposure modulate IgE‐ and non‐IgE‐dependent basophil activation. Immunology, 162(1), 92-104.

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Quantifying Lung Inflammatory Cytokines

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Lung IL-1β (R&D Systems), IL-25 and IL-33 (Thermo Fisher Scientific) were measured by ELISA. ELISA data were analyzed by BioTek Gen5 software (Winooski, VT). Total lung protein concentration was measured by BCA protein assay (Thermo Fisher Scientific).

Han M., Ishikawa T., Bermick J.R., Rajput C., Lei J., Goldsmith A.M., Jarman C.R., Lee J., Bentley J.K, & Hershenson M.B. (2020). IL-1β prevents ILC2 expansion, type 2 cytokine secretion and mucus metaplasia in response to early-life rhinovirus infection in mice. Allergy, 75(8), 2005-2019.

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