Recombinant human il 23

Manufactured by R&D Systems

Sourced in United States

Recombinant human IL-23 is a cytokine that belongs to the IL-12 family. It is composed of two subunits, p19 and p40. IL-23 plays a role in the maintenance and expansion of Th17 cells.

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Spelling variants of this product

IL-23 (200 citations) Recombinant IL-23 (12 citations) Human IL-23 (3 citations)

8 protocols using recombinant human il 23

Th17 Cell Induction from PBMCs

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Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using Lymphocyte Separation Media (Accurate Chemical and Scientific, Westbury, NY, USA) and buoyant density centrifugation. PMBC stimulation was performed by seeding 48-well, flat-bottom, cell culture plates with 500,000 PBMCs ± 1 × 10⁶ heat-killed (HK) C. albicans (prepared by boiling ~4 × 10⁸C. albicans cells for 45 minutes) or a Th17 differentiating cocktail of recombinant human IL-1β (10 ng/ml), recombinant human IL-6 (50 ng/ml), recombinant human IL-23 (20 ng/ml), recombinant human transforming growth factor beta (TGFβ; 10 ng/ml), recombinant human IL-2 (24 IU/ml), anti-IL-12 (5 μg/ml) and anti-IL-4 (5 μg/ml) (R&D Systems, Minneapolis MN, USA)
[20 (link)]. Supernatants were collected after 5 days and were analyzed in triplicate for IL-17A by enzyme-linked immunosorbent assay (eBiosciences, San Diego, CA, USA). C. albicans was prepared by culturing strain CAF2-1 in yeast peptone dextrose at 30°C overnight with agitation.

Bishu S., Su E.W., Wilkerson E.R., Reckley K.A., Jones D.M., McGeachy M.J., Gaffen S.L, & Levesque M.C. (2014). Rheumatoid arthritis patients exhibit impaired Candida albicans-specific Th17 responses. Arthritis Research & Therapy, 16(1), R50.

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T Cell Activation and Cytokine Assay

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Cells were cultured in DMEM or IMDM medium (Gibco) supplemented as described (31 (link)) at 37°C/10% CO₂. For ex vivo activation of FACS-sorted naïve and memory T cell subsets, 2–3 × 10⁴ T cells were added to round-bottom tissue culture treated plates (Corning) together with anti-CD3/anti-CD28-coated beads (Invitrogen; 3 beads:1 cell), and recombinant human IL-2 (BD Bioscience; 10 Units/mL). recombinant human IL-23 (R&D Systems; 20 ng/mL) was added as indicated. For intracellular cytokine staining, cultured cells were washed once in complete medium and resuspended in medium containing PMA (10 nM), Ionomycin (1 μM) and Brefeldin A (10 μg/ml) for 3–4 hr.

Carlson T.J., Pellerin A., Djuretic I.M., Trivigno C., Koralov S.B., Rao A, & Sundrud M.S. (2014). Halofuginone-induced amino acid starvation regulates Stat3-dependent Th17 effector function and reduces established autoimmune inflammation. Journal of immunology (Baltimore, Md. : 1950), 192(5), 2167-2176.

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Phenotyping Intestinal Immune Cells in CD

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Single-cell suspensions isolated from intestinal tissues of CD patients were stained with FITC-conjugated anti-human lineage cocktail 3 (Lin3) (CD3, CD14, CD19, and CD20; clone: MφP9, L27, SK7, and SJ25C1; BD Biosciences), PerCP-conjugated anti-human CD45 (clone: HI30, BioLegend), APC-conjugated anti-human CD127 (clone: A019D5, BioLegend), PE-conjugated anti-human NKp44 (clone: P44-8, BioLegend), PE-Cy7-conjugated anti-human CD117 (clone: 104D2, eBioscience), Brilliant Violet 421-conjugated anti-human CRTH2 (clone: BM16, BioLegend), and Alexa Fluor 647-conjugated anti-STAT3 Phospho-Tyr705 (clone: 13A3-1, BioLegend). Viability was assessed by Fixable Yellow Dead Cell Stain (Thermo Fisher Scientific). For phosphoflow staining, cells were stimulated with 20 ng/ml of recombinant human IL-23 (carrier free, R&D) for 15 mins and were permeabilized by True-Phos™ Perm Buffer (BioLegend) following the manufacturer's protocol. AbC™ Total Antibody Compensation Beads (Cat. No. A10497, Life Technologies) and ArC™ Amine Reactive Compensation Beads (Cat. No. A10346, Life Technologies) were used for compensation. Data were acquired on an LSRFortessa™ flow cytometer (BD Biosciences) using BD FACSDiva™ software in the Cytometry Core at the University of Florida and analyzed by FlowJo software (Version 10, Tree Star Inc.).

Tang Y., Tan S.A., Iqbal A., Li J, & Glover S.C. (2019). STAT3 Genotypic Variant rs744166 and Increased Tyrosine Phosphorylation of STAT3 in IL-23 Responsive Innate Lymphoid Cells during Pathogenesis of Crohn's Disease. Journal of Immunology Research, 2019, 9406146.

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Stimulation and Analysis of cTh17 Cells

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FACS-purified cTh17 cells [CD4⁺IL-7R⁺CD25^lo/-CCR6⁺CXCR3^-CCR5[-]] from buffy-coated blood of healthy donors [HD] were stimulated in plate-bound anti-CD3 [0.02 μg/mL, BD Biosciences] and anti-CD28 antibodies [6 μg/mL, BD Biosciences] in complete RMPI medium [5% human serum, 1 mmol/L sodium pyruvate, 10 mmol/L nonessential amino acids, 1% penicillin/streptomycin] in the presence or absence of recombinant human IL-23 [10 ng/mL, R&D Systems], recombinant human IL-12 [10 ng/mL, R&D Systems], IL-1β, IL-6, TNF [10ng/ml, R&D Systems], or neutralising anti-TNF antibodies [Ebioscience]. After 4 days, cells were transferred to uncoated plates and cultured for an additional 2–6 days under the same cytokine conditions, and then analysed for CXCR3 or CCR5 surface expression.

Paroni M., Leccese G., Ranzani V., Moschetti G., Chiara M., Perillo F., Ferri S., Clemente F., Noviello D., Conforti F.S., Ferrero S., Karnani B., Bosotti R., Vasco C., Curti S., Crosti M.C., Gruarin P., Rossetti G., Conte M.P., Vecchi M., Pagani M., Landini P., Facciotti F., Abrignani S., Caprioli F, & Geginat J. (2023). An Intestinal Th17 Subset is Associated with Inflammation in Crohn’s Disease and Activated by Adherent-invasive Escherichia coli. Journal of Crohn's & Colitis, 17(12), 1988-2001.

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γδT Cell Activation by Tumor Factors

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γδT cells isolated from normal tissues were cultured in the conditional medium containing heat-killed E. coli, supernatants derived from normal tissues (NS), supernatants derived from tumor tissues (TS), or recombinant human IL-23 (10 μg/ml, R&D Systems). IL-23 neutralizing antibody (Clone 24901, 0.1μg/ml,) was added when needed. At day 14, IL-17⁺ cells were detected by FCM.

Wu P., Wu D., Ni C., Ye J., Chen W., Hu G., Wang Z., Wang C., Zhang Z., Xia W., Chen Z., Wang K., Zhang T., Xu J., Han Y., Zhang T., Wu X., Wang J., Gong W., Zheng S., Qiu F., Yan J, & Huang J. (2014). γδT17 Cells Promote the Accumulation and Expansion of Myeloid-Derived Suppressor Cells in Human Colorectal Cancer. Immunity, 40(5), 785-800.

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In Vitro T-Cell Polarization

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Naive and memory T-cell subsets (10⁶ cells/ml) sorted by FACS were stimulated with immobilized CD3 and soluble CD28 Abs (1 μg/ml) and cultured in RPMI 1640 media supplemented with human recombinant IL-23 (50 ng/ml), TGF-β (10 ng/ml), IL-1β (10 ng/ml), and IL-6 (50 ng/ml) cytokines and neutralizing IL-4 (1 μg/ml) and IFN-γ Abs (10 μg/ml) (R&D Systems) for 12 days. Media including polarizing cytokines, Abs, and IL-2 (5 ng/ml) was refreshed at day 4 and 8 post-culture. Cells were split at day 4 and/or 8 post-culture for an optimal density 1-2x10⁶ cells/well.

DaFonseca S., Niessl J., Pouvreau S., Wacleche V.S., Gosselin A., Cleret-Buhot A., Bernard N., Tremblay C., Jenabian M.A., Routy J.P, & Ancuta P. (2015). Impaired Th17 polarization of phenotypically naive CD4+ T-cells during chronic HIV-1 infection and potential restoration with early ART. Retrovirology, 12, 38.

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Pathogenic Th17 Cell Differentiation in Rheumatoid Arthritis

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PBMC from RA patients were stimulated with MoAbs against CD3/CD28 (5.0 ug/ml in both cases, plate-bound) in IMDM culture medium (Gibco, Grand Island, NY), supplemented with 10% fetal bovine serum, glutamine (2.0 mM), and penicillin (100 u/ml)/streptomycin (100 μg/ml), and incubated at 5% CO₂ and 37°C for 6 days. In order to induce the differentiation of pathogenic Th17 cells, human recombinant IL-23 (R&D Systems, Minneapolis, MN, 10.0 ng/ml, or this cytokine plus IL-1β (Peprotech, Rocky Hill, NJ, 8.0 ng/ml) was added, at days 1 and 3 of cell culture. Three hours before harvesting, cells were incubated with the leukocyte activation cocktail (PMA plus Ionomycin) and GolgiPlug (BD Pharmingen), and then cells were stained and analyzed by flow cytometry, as described above.

Vitales-Noyola M., Hernández-Castro B., Alvarado-Hernández D., Baranda L., Bernal-Silva S., Abud-Mendoza C., Niño-Moreno P, & González-Amaro R. (2022). Levels of Pathogenic Th17 and Th22 Cells in Patients with Rheumatoid Arthritis. Journal of Immunology Research, 2022, 5398743.

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Quantification of Phosphorylated STAT3

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Kit225 cells were seeded in 384-well plate at a density of 1×10⁵ cells/well in 4 µL HBSS, and incubated for 2 hours in a humidified, 5% CO₂ cell culture incubator at 37°C. The cells were treated with serially diluted compounds for 1 hour and stimulated with 100 ng/mL human recombinant IL23 (R&D Systems) for 20 minutes. The treated cells were then lysed and cellular phosphorylated-STAT3 (Tyr705) levels were measured by AlphaLISA (PerkinElmer, Cat# ALSU-PST3) according to the manufacturer’s instructions. Inhibition data were calculated by comparison to vehicle control wells for 0% inhibition and non-stimulated control wells for 100% inhibition. Dose response curves were then generated to determine the concentration required to suppress 50% of cellular response (IC₅₀) as derived by non-linear regression analysis using GraphPad Prism.

Zhou Y., Li X., Shen R., Wang X., Zhang F., Liu S., Li D., Liu J., Li P., Yan Y., Dong P., Zhang Z., Wu H., Zhuang L., Chowdhury R., Miller M., Issa M., Mao Y., Chen H., Feng J., Li J., Bai C., He F, & Tao W. (2022). Novel Small Molecule Tyrosine Kinase 2 Pseudokinase Ligands Block Cytokine-Induced TYK2-Mediated Signaling Pathways. Frontiers in Immunology, 13, 884399.

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