Rhil 23

Manufactured by R&D Systems

Sourced in United States

RhIL-23 is a recombinant human Interleukin-23 protein. Interleukin-23 is a cytokine that plays a role in the regulation of immune and inflammatory responses.

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12 protocols using rhil 23

CD133+ Cancer Stem-like Cells Sphere Formation

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CD133⁺CSLCs with or without rhIL-23 stimulation were seeded at concentrations of 1, 10, 100 and 1000 cells per well on 96-well plates. Then plates were incubated at 37°C in a humid incubator with 5% CO2 for 2 weeks, spheres containing ≥3 cells were counted under an inverted microscope. During the sphere formation assay, cells were treated with IL-23 neutralizing antibody (R&D Systems), IL-23 inhibitor STA-5326 (Toronto Research Chemicals Inc.), rhIL-23 (R&D Systems), STAT3 inhibitor Cucurbitacin (0.2μM) and p65 inhibitor PDTC (0.5μM).

Wang D., Xiang T., Zhao Z., Lin K., Yin P., Jiang L., Liang Z, & Zhu B. (2016). Autocrine interleukin-23 promotes self-renewal of CD133+ ovarian cancer stem-like cells. Oncotarget, 7(46), 76006-76020.

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Cytokine and Immune Cell Profiling

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IL-4 (BD555194), IL-10 (DY217b), IFN-γ (DY285), IL-13 (DY213), IL-17 (DY317), IL-22 (DY782), IL-26 (CSB-E11716h), rhIL-26 (R&D), rhIL-23 (R&D), rhIL-2 (R&D), Blocking mouse anti human HLA-Class I (W6/32) and anti-HLA-C (Abcam) both used at 10μg/ml, anti-MHC-II (MS163P1ABX) (Fisher), anti-IL-26 mAb1375, clone 197505 (R&D), CD4 (OKT4) (BD), IL-17PE (BD), IFN-γ- FITC (BD), CD14 FITC (BD) LL-37 (Innovagen). Mouse IgG1 and IgG2b isotype matched antibodies were used as controls.

Agak G.W., Kao S., Ouyang K., Qin M., Moon D., Butt A, & Kim J. (2017). Phenotype and antimicrobial activity of Th17 cells induced by Propionibacterium acnes strains associated with healthy and acne skin. The Journal of investigative dermatology, 138(2), 316-324.

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Cell Culture Conditions and Stimulation Assays

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HEK293T cells were cultured in DMEM (Gibco) supplemented with 10% FBS (Invitrogen). EBV-B cells were cultured in RPMI (Gibco) supplemented with 10% FBS. HVS-T cells were cultured in a 1:1 mixture (by volume) of RPMI and Panserin 401 (PAN Biotech) supplemented with 10% FBS, GlutaMAX (350 µg/ml; Gibco), gentamicin (0.1 mg/ml; Gibco), and rhIL-2 (20 IU/ml; Roche).
The cells were starved for 2 h by incubation in serum-free RPMI. The cells were then left unstimulated or were stimulated with rhIFN-α 2b (10⁵ IU/ml; Schering), rhIL-23 (100 ng/ml; R&D Systems), rhIL-12 (20 ng/ml; R&D Systems), or rhIL-10 (50 ng/ml; PeproTech) for 5 min to assess the phosphorylation of JAKs, and for 30 min to assess the phosphorylation of STATs. For quantitative RT-PCR, cells were stimulated for 6 h with IL-10 (50 ng/ml) or IFN-α (10⁵ IU/ml). For RNA-seq experiments, cells were starved for 1.5 h in RPMI containing 1% FCS and were stimulated for 2 h with IFN-α (10⁵ IU/ml) and IL-21 (100 ng/ml). RNA was extracted with the Zymoresearch kit. For scRNA-seq experiments, PBMCs were either left unstimulated or were stimulated with 100 ng/ml of IL-23 or 10³ IU/ml IFN-α2b for 6 h.

Ogishi M., Arias A.A., Yang R., Han J.E., Zhang P., Rinchai D., Halpern J., Mulwa J., Keating N., Chrabieh M., Lainé C., Seeleuthner Y., Ramírez-Alejo N., Nekooie-Marnany N., Guennoun A., Muller-Fleckenstein I., Fleckenstein B., Kilic S.S., Minegishi Y., Ehl S., Kaiser-Labusch P., Kendir-Demirkol Y., Rozenberg F., Errami A., Zhang S.Y., Zhang Q., Bohlen J., Philippot Q., Puel A., Jouanguy E., Pourmoghaddas Z., Bakhtiar S., Willasch A.M., Horneff G., Llanora G., Shek L.P., Chai L.Y., Tay S.H., Rahimi H.H., Mahdaviani S.A., Nepesov S., Bousfiha A.A., Erdeniz E.H., Karbuz A., Marr N., Navarrete C., Adeli M., Hammarstrom L., Abolhassani H., Parvaneh N., Al Muhsen S., Alosaimi M.F., Alsohime F., Nourizadeh M., Moin M., Arnaout R., Alshareef S., El-Baghdadi J., Genel F., Sherkat R., Kiykim A., Yücel E., Keles S., Bustamante J., Abel L., Casanova J.L, & Boisson-Dupuis S. (2022). Impaired IL-23–dependent induction of IFN-γ underlies mycobacterial disease in patients with inherited TYK2 deficiency. The Journal of Experimental Medicine, 219(10), e20220094.

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IL-23 Stimulation of Alveolar Macrophages

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After removal of nonadherent cells from the lavage fluid, cultured AM containing small numbers of lymphocytes from both HIV1⁺ and HIV1⁻ individuals were incubated with RPMI media in 12-well plates at 37°C in a 5% CO₂ humidified incubator. AM were treated with serum-free media alone or with serum-free media containing rhIL-23 (R&D systems) at various concentrations. Treated AM/lymphocytes were cultured overnight for quantitative cDNA analysis or 48 hours for the analysis of substrate (MMP-9) concentration in conditioned media.

Barjaktarevic I.Z., Crystal R.G, & Kaner R.J. (2016). The Role of Interleukin-23 in the Early Development of Emphysema in HIV1+ Smokers. Journal of Immunology Research, 2016, 3463104.

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Th17 Differentiation from Naive T Cells

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Human cord blood samples were obtained from the Department of Prenatal Medicine and Midwifery of the Medical School Hannover (MHH). All work with human blood samples was approved by the local ethics committee and informed consent was obtained from all subjects. After Ficoll (Biocoll) gradient, naive CD44⁺ T cells were enriched by magnetic separation using the EasySep™ Human Naive CD4⁺ T Cell Isolation Kit (Stemcell Technologies). 5×10⁴ T cells were cultured for 6 days in the presence of plate-bound aCD3ε (5 mg mL^–1), in X-Vivo 15 medium (Lonza), supplemented with 2% heat-inactivated FCS (Biochrom), 500 U penicillin-streptomycin (PAA laboratories), and 50 mM β-mercaptoethanol (Life Technologies). To polarize the cells towards a Th17 cell phenotype, the medium was supplemented further with rhIL-1β (10 ng mL^–1, R&D Systems), rhIL-23 (20 ng mL^–1, R&D Systems), rhIL-6 (20 ng mL^–1; Peprotech), rhIL-21 (20 ng mL^–1; Peprotech), rhTGF-β1 (3 ng mL^–1; Peprotech), aCD28 (500 ng mL^–1), and 20 mM NaCl. Arg C and Linezolid were added at the indicated concentrations from day 0.

Almeida L., Dhillon-LaBrooy A., Castro C.N., Adossa N., Carriche G.M., Guderian M., Lippens S., Dennerlein S., Hesse C., Lambrecht B.N., Berod L., Schauser L., Blazar B.R., Kalesse M., Müller R., Moita L.F, & Sparwasser T. (2021). Ribosome-Targeting Antibiotics Impair T Cell Effector Function and Ameliorate Autoimmunity by Blocking Mitochondrial Protein Synthesis. Immunity, 54(1), 68-83.e6.

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Th17 Cell Differentiation from Naïve T Cells

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Human cord blood samples were obtained from the Department of Prenatal Medicine and Midwifery of the Medical School Hannover (MHH). All work with human blood samples was approved by the local ethics committee and informed consent was obtained from all subjects. After Ficoll (Biocoll) gradient, naive CD44⁺ T cells were enriched by magnetic separation using the EasySep^™ Human Naïve CD4⁺ T Cell Isolation Kit (Stemcell Technologies). 5×10⁴ T cells were cultured for 6 days in the presence of plate-bound aCD3ε (5 mg mL⁻¹), in X-Vivo 15 medium (Lonza), supplemented with 2% heat-inactivated FCS (Biochrom), 500 U penicillin-streptomycin (PAA laboratories), and 50 mM β-mercaptoethanol (Life Technologies). To polarize the cells towards a Th17 cell phenotype, the medium was supplemented further with rhIL-1β (10 ng mL⁻¹, R&D Systems), rhIL-23 (20 ng mL⁻¹, R&D Systems), rhIL-6 (20 ng mL⁻¹; Peprotech), rhIL-21 (20 ng mL⁻¹; Peprotech), rhTGF-β1 (3 ng mL⁻¹; Peprotech), aCD28 (500 ng mL⁻¹), and 20 mM NaCl. Arg C and Linezolid were added at the indicated concentrations from day 0.

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Differentiation of Naïve CD4+ T Cells into Th17 Cells

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Human peripheral blood mononuclear cells (PBMCs) were isolated from the buffy coats of participants by Ficoll-paque Plus (GE Healthcare catalog #17-1440-02). Naïve CD4+ T cells were isolated from the PBMCs using a naïve T cell isolation kit (Miltenyi Biotec catalog # 130-094-131) per manufacturer's instruction. CD4+ naïve T cells were activated using an activation/expansion kit (Miltenyi Biotec catalog #130-091-441) with anti-CD3 and anti-CD28 bound to bead particles at the ratio of 1 bead particle per 2 cells. CD4+ T cells were cultured differentiated to become Th17 cells with hIL-2 (10ng/ml), rhIL-1β (10ng/ml), rhTGF-β (1ng/ml), rhIL-6 (10ng/ml), rhIL-23 (10ng/ml), anti-IFN-γ (10μg/ml), and/or anti-IL-4 (10μg/ml) in T cell culture media. T cell culture media was RPMI containing 10% FBS, 1% penicillin/streptomycin, 2mM l-glutamine, 10mM HEPES, 0.1mM non-essential amino acids, and 1mM sodium pyruvate (Gibco, Carlsbad, CA). All antibodies and rhIL-1β, rhIL-6, and rhIL-23 were purchased from R&D Systems. rhIL-2 and rhTGF-β was purchased from PeproTech (Rocky Hill, NJ).

Newcomb D.C., Cephus J.Y., Boswell M.G., Fahrenholz J.M., Langley E.W., Feldman A.S., Zhou W., Dulek D.E., Goleniewska K., Woodward K.B., Sevin C.M., Hamilton R.G., Kolls J, & Peebles RS J.r. (2015). Estrogen and progesterone decrease Let-7f miRNA expression and increase IL-23/IL-23R signaling and IL-17A production in severe asthma. The Journal of allergy and clinical immunology, 136(4), 1025-1034.e11.

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Comprehensive Immune Cell Profiling

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Fluorochrome-labeled mAb (all, Becton-Dickinson, Franklin Lakes, New Jersey, USA) were used for surface staining: CD3-FITC (clone SK7), CD4-Alexa Fluor 700 (RPA-T4), CD25-PE (2A3), CD45-V450 (HI30), CD127-BV650 (HIL-7R-M21), CD161-APC (DX12), CD196-PerCPCy5.5 (IIA9), and TCRγδ-BV605 (B1). The vital stain, Live/Dead Aqua, was purchased from Invitrogen/Thermo Fisher Scientific (Waltham, MA; catalog No. L34957). Recombinant human (rh) cytokines were purchased from BD Biosciences, San Jose, CA (rhIL-2) , Peprotech, Inc., Rocky Hills, New Jersey, USA (rhIL-1β, rhIL-6), and R&D Systems, Minneapolis, Minnasota, USA (rhIL-23, rhTGFβ, rhS100A12).

Lufkin T., Lohnes D., Mark M., Dierich A., Gorry P., Gaub M.P., LeMeur M, & Chambon P. (1993). High postnatal lethality and testis degeneration in retinoic acid receptor alpha mutant mice.. Proceedings of the National Academy of Sciences of the United States of America, 90(15), 7225-7229.

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Quantifying IL-22 Production in ILC3s

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To measure IL-22 production, HSPC-ILC were incubated for 5 h (flow cytometry) or 24 h (ELISA) with the following ILC3 stimulatory cytokines: 50 ng/mL rhIL-1b (R&D systems, 201-LB-025), 20 U/mL rhIL-2 (Novartis, 709406), 50 ng/mL rhIL-7, 50 ng/mL rhIL-23 (R&D systems, 1290-IL-010/CF); 25 ng/mL phorbol 12-myristate 13-acetate (PMA, Abcam, ab147465), and 1 mg/mL ionomycin (Sigma, I9657-1MG). After 1 h stimulation, 1 mL GolgiPlug (BD, 555029) per mL medium was added to the flow cytometry samples. After 5 h stimulation, flow cytometry samples were incubated with nanogam, fixable live/dead marker near-IR and the following antibodies: CD56-BV510, CD94-FITC, CD117-PE-Cy7 and CD336(NKp44)-APC. Next, cells were fixed, permeabilized and intracellularly stained with anti-IL-22-PE (R&D systems, IC7821P, RRID:AB_495011). After 24 h stimulation, supernatants were evaluated for IL-22 secretion following manufacturer's instructions (R&D systems, DY782).

Van der Meer J.M.R., Bulder I., Kuijk C., Kleijer M., Verheij M.W., Omar S.Z., Haverkate N.J.E., Dolstra H., Blom B., Hazenberg M.D, & Voermans C. (2024). Generation of human ILC3 from allogeneic and autologous CD34⁺ hematopoietic progenitors toward adoptive transfer. Cytotherapy, 26(2).

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Differentiation of Human T-cell Subsets

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The differentiation of human T-cell subsets from naive CD4⁺ T cells was performed as previously described.52 53 (link) Brieﬂy, the treated DCs were puriﬁed and co-cultured with human naive CD4⁺ T cells at a ratio of 1:10 in plate-bounded anti-CD3/CD28 (2 µg/mL, Bio X Cell) in normal T-cell culture medium for 6 days. For Th1 differentiation, DCs and naive T cells were co-cultured at a ratio of 1:10 in the presence of anti-IL-4-neutralizing antibody (10 µg/mL, 11B11; Bio X Cell) and recombinant human (rh)IL-12 (10 ng/mL; R&D Systems). For Th17 differentiation, DCs and naive T cells were co-cultured at a ratio of 1:10 in the presence of rhIL-1β (10 ng/mL), rhIL-6 (20 ng/mL), rhIL-23 (10 ng/mL) and recombinant human transforming growth factor-beta (rhTGF-β) (3 ng/mL) (R&D System) for 6 days.

Si F., Liu X., Tao Y., Zhang Y., Ma F., Hsueh E.C., Puram S.V, & Peng G. (2024). Blocking senescence and tolerogenic function of dendritic cells induced by γδ Treg cells enhances tumor-specific immunity for cancer immunotherapy. Journal for Immunotherapy of Cancer, 12(4), e008219.

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