Il 36β

Manufactured by R&D Systems

Sourced in United States

IL-36β is a recombinant human protein that belongs to the IL-1 cytokine family. It is a secreted protein involved in the regulation of immune and inflammatory responses.

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4 protocols using il 36β

Murine T Cell Activation and Differentiation

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For in vitro cultures, naïve CD3⁺, CD4⁺, CD8⁺ T cells from spleen and lymph nodes (inguinal, brachial, axillary and cervical) of male and female C57BL/6J mice (6–9 weeks old) were purified by negative selection using Dynabeads™ Untouched™ Mouse Cells Kits from ThermoFisher Scientific (Waltham, MA). Cells were then differentiated in vitro using Dynabeads™ Mouse T-Activator CD3/CD28 for T cell Expansion and Activation from ThermoFisher Scientific (1:1 ratio) (Waltham, MA), plus hrIL-2 (30 U/ml), in the presence or absence of NAM 30 mM or 10 mM. After 66 h live cells were recovered using Lympholyte®-M from Cederlane (Burlington, NC) as per manufacturer’s instructions. Lastly, cells were restimulated using either Dynabeads™ Mouse T-Activator CD3/CD28 or PMA + ionomicyn (PMAi). For ex-vivo cultures, in vivo–primed CD8⁺ T cells from spleen and lymph nodes (inguinal, brachial, axillary and cervical) were purified by negative selection using Dynabeads™ Untouched™ Mouse CD8 Cells Kit from ThermoFisher Scientific (Waltham, MA) following biotinylated α-CD45.2 Ab from ThermoFisher Scientific (Waltham, MA). Cells were then restimulated using SIINFEKL, PMAi or IL-2/IL-12 plus IL-33 or IL-36β (R&D Systems, Minneapolis, MN) for the indicated time points.

Agliano F., Karginov T.A., Ménoret A., Provatas A, & Vella A.T. (2022). Nicotinamide breaks effector CD8 T cell responses by targeting mTOR signaling. iScience, 25(3), 103932.

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Inflammatory Cytokine and Cigarette Smoke Exposure Protocol

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Recombinant IL-1α, IL-36α, IL-36β, IL-36γ, IL-36Ra, TNF-α, neutrophil elastase, cathepsin G, proteinase-3, and anti–IL-36γ antibody (catalog AF2320) were purchased from R&D Systems. Rabbit anti–goat Ig/HRP (P0449) was purchased from Agilent. Poly(I:C) was purchased from Sigma-Aldrich. Nontypeable H. influenzae were obtained from the National Collection of Type Cultures (strain no. 1269) and were heat-killed by incubation at 65°C for 10 minutes as described previously (55 (link)). CSE was generated as previously described (56 (link)) from full-strength Marlboro cigarette (Phillip Morris). Budesonide was purchased from Thermo Fisher Scientific.

Baker J.R., Fenwick P.S., Koss C.K., Owles H.B., Elkin S.L., Fine J., Thomas M., El Kasmi K.C., Barnes P.J, & Donnelly L.E. (2022). IL-36 receptor agonist and antagonist imbalance drives neutrophilic inflammation in COPD. JCI Insight, 7(15), e155581.

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Periostin secretion in mouse fibroblasts

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Mouse embryonic fibroblasts were cultured in DMEM supplemented with 10% FCS at 1 × 10⁵ cells/ml overnight. The medium was then replaced with Opti-MEM I (Life Technologies) and stimulated with mouse recombinant IL-17 (Sigma-Aldrich), IL-21, IL-22, IL-36β (R&D Systems, Minneapolis, MN, USA), or IL-13 (PeproTech, Rocky Hill, NJ, USA) at 50 ng/ml for 24 h. The ELISA analysis for periostin in the supernatants was performed as described previously.^{14 (link)}

Arima K., Ohta S., Takagi A., Shiraishi H., Masuoka M., Ontsuka K., Suto H., Suzuki S., Yamamoto K.I., Ogawa M., Simmons O., Yamaguchi Y., Toda S., Aihara M., Conway S.J., Ikeda S, & Izuhara K. (2014). Periostin contributes to epidermal hyperplasia in psoriasis common to atopic dermatitis. Allergology international : official journal of the Japanese Society of Allergology, 64(1), 41-48.

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Splenocyte Cytokine Profiling and Tumor ELISA

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After RBC lysis, 0.5−1 × 10⁶ splenocytes were seeded in a 96-well flat-bottom plate in a final volume of 200 μl. For peptide stimulation, cells were treated with 5 μg/ml Eα (Invivogen, San Diego, CA) or 1X PMA + I (Fisher Scientific, Hampton, NH) in the presence of 5 μg/ml BFA (Calbiochem). Cells were further treated with Y-Ae – biotin or mouse IgG2b – biotin control antibody (eBioscience, Waltham, MA), and then harvested after 4 h. Cytokine stimulations were performed in the absence of BFA with the following cytokine concentrations: 50 U/ml IL-2, 2.5 ng/ml IL-12, 1 ng/ml IL-33, and 10 ng/ml IL-36β (R&D Systems, Minneapolis, MN). The following morning, cells were treated with 5 μg/ml BFA for 4 h, and then harvested for analysis via flow cytometry.
IL-12 (p70) and IL-33 levels in B16 tumor lysates were measured using ELISA kits from BD Bioscience and R&D Systems, respectively.

Del Valle C.M., Maxwell J.R., Xu M.M., Menoret A., Mittal P., Tsurutani N., Adler A.J, & Vella A.T. (2019). Costimulation Induces CD4 T Cell Antitumor Immunity via an Innate-like Mechanism. Cell reports, 27(5), 1434-1445.e3.

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