Il 6r

Manufactured by R&D Systems

Sourced in United States

The IL-6R is a recombinant human protein that functions as the receptor for the cytokine Interleukin-6 (IL-6). It is a member of the type I cytokine receptor family and plays a crucial role in the regulation of immune and inflammatory responses.

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Lab products found in correlation

Interleukin 6 (il 6), by R&D Systems (3 mentions) Biacore 3000, by Cytiva (1 mentions) Antigen unmarking solution, by Vector Laboratories (1 mentions) Keratin 5, by BioLegend (1 mentions) Cytokeratin 8, by BioLegend (1 mentions) Pannoramic 250 flash 3, by 3DHISTECH (1 mentions) Tunel assay kit, by Abcam (1 mentions) Pannoramic viewer software, by 3DHISTECH (1 mentions) Human il 6, by R&D Systems (1 mentions) Human tnf α, by R&D Systems (1 mentions) Human ifn γ, by R&D Systems (1 mentions) Egm 2mv bulletkit, by Cambrex (1 mentions) Phorbol myristate acetate (pma), by R&D Systems (1 mentions) Hdmec, by Lonza (1 mentions) Lipopolysaccharides (lps), by Merck Group (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Nrk 49f, by American Type Culture Collection (1 mentions) Dulbecco modified eagle medium (dmem), by Wisent (1 mentions) Pd98059, by Santa Cruz Biotechnology (1 mentions) Ly294002, by R&D Systems (1 mentions)

Spelling variants of this product

Anti-IL-6R (5 citations) Human IL-6R alpha Quantikine ELISA Kit (4 citations) Soluble IL-6R (3 citations) Mouse IL-6R alpha DuoSet ELISA (2 citations) Anti-human IL-6R antibody (2 citations) IL-6R antibody (2 citations) IL-6R alpha antibody (2 citations) Human IL-6R alpha DuoSet ELISA kit (2 citations) Anti-IL-6R antibody (2 citations) IL-6+sIL-6R (2 citations)

9 protocols using il 6r

Characterizing IL-6R Binding Interactions

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Competition ELISA was performed as follows: non-neutralizing IL-6R antibody (BN12; Diaclone) was immobilized on Maxisorp plate at a concentration of 1 μg/ml overnight at 4 °C. Then 0.1 μg/ml of IL-6R (R&D Systems) was incubated 1 h at room temperature. Biotinylated human IL-6 (0.025 μg/ml) alone or in combination with a concentration series of antibody was added for 1 h at room temperature. After washing, biot-IL-6 bound to the IL-6R was detected with Strep-HRP. After addition of TMB and H₂SO₄ to stop the reaction, optical density was read at 450 nm. IL-6 was biotinylated using the Pierce kit with the modification that the biotinylation reaction was performed at pH 5.5 to only biotinylate the N terminus of IL-6. Antibody competition with biot-IL-6 for IL-6R binding was expressed as a percentage of biot-IL-6 binding as compared with biot-IL-6 alone using GraphPad Prism v6.
Surface plasmon resonance (SPR; Biacore 3000) was used for competition experiments on a low density IL-6 coating (∼75–100 resonance units). mAb 61H7 was first injected at 50 μg/ml with a flow rate of 30 μl/min. A second antibody (50 μg/ml of 61H7 or 68F2) was added using COINJECT procedure at the same flow rate to investigate competition for binding to coupled IL-6.

Blanchetot C., De Jonge N., Desmyter A., Ongenae N., Hofman E., Klarenbeek A., Sadi A., Hultberg A., Kretz-Rommel A., Spinelli S., Loris R., Cambillau C, & de Haard H. (2016). Structural Mimicry of Receptor Interaction by Antagonistic Interleukin-6 (IL-6) Antibodies. The Journal of Biological Chemistry, 291(26), 13846-13854.

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Comprehensive Immunohistochemistry Profiling

Cited 1 time

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Immunohistochemistry was performed as previously described (17 (link)). A pressure cooker (95°C for 30 min followed by 120°C for 10 s) was used for antigen retrieval using Antigen Unmarking Solution (Vector Laboratories). Antibodies specific to CHD1 (Sigma, #HPA022236), AKT-473P (Cell Signaling, #4060s), Keratin 5 (BioLegend 905501), Cytokeratin-8 (BioLegend 904801), CD8 (Bioss bs-0648R), Ly6G (BioLegend 127601), CD15 (DAKO M3631), IL‑6R (R&D Systems AF1830-SP) and Ki67 (Thermo Scientific RM 9106-S1) were purchased. TUNEL staining was performed using the TUNEL Assay Kit (Abcam ab206386) according to manufacturer’s instructions. Slides were scanned using Pannoramic 250 Flash III (3DHISTECH Ltd) and images were captured through Pannoramic Viewer software (3DHISTECH Ltd). Human prostate hyperplasia and cancer tissue array samples were purchased from US Biomax (PR753a).

Zhao D., Cai L., Lu X., Liang X., Li J., Chen P., Ittmann M., Shang X., Jiang S., Li H., Meng C., Flores I., Song J.H., Horner J.W., Lan Z., Wu C.J., Li J., Chang Q., Chen K.C., Wang G., Deng P., Spring D.J., Wang Y.A, & DePinho R.A. (2020). Chromatin Regulator, CHD1, Remodels the Immunosuppressive Tumor Microenvironment in PTEN-Deficient Prostate Cancer. Cancer discovery, 10(9), 1374-1387.

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Endothelial Cell Culture and Activation

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HUVECs were derived and propagated as described previously (31 (link)). HDMECs (Lonza) were maintained in endothelial basal medium-2 (EBM-2) supplemented with endothelial growth medium-2 microvascular (EGM-2MV BulletKit, Clonetics). BAF-130 cells (murine BAF-B03 cells stably expressing human gp130) were previously reported (27 (link)). PBMCs were obtained from volunteer donors according to an approved protocol. Cells were stimulated with PMA (10 ng/ml; R&D Systems) or LPS (1 μg/ml; Sigma) or with human TNF-α (20 ng/ml; R&D Systems) and human IFN-γ (1 or 10 ng/ml; R&D Systems) for 24 hours in complete culture medium. To activate gp130-pSTAT3 signaling, HUVECs were stimulated (0 to 45 min) with p19 (1 μg/ml; Abnova) or with human IL-6 (200 to 1000 ng/ml; R&D Systems) and IL-6R (200 to 2500 ng/ ml; R&D Systems). In selected experiments, IL-6 and IL-6R were used to preactivate HUVECs for 18 to 24 hours. For production of serum-free cell culture medium, endothelial cells were cultured in serumfree M199 complete medium for 24 to 72 hours. TCA was used to precipitate proteins from the culture medium. To reduce background activation, HUVECs were cultured for 18 hours in M199 medium containing 0.5% bovine serum albumin (BSA).

Espígol-Frigolé G., Planas-Rigol E., Ohnuki H., Salvucci O., Kwak H., Ravichandran S., Luke B., Cid M.C, & Tosato G. (2016). Identification of IL-23p19 as an endothelial proinflammatory peptide that promotes gp130-STAT3 signaling. Science signaling, 9(419), ra28.

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Renal Fibroblast Response to Cytokines

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Renal fibroblast cell line NRK-49F was from American Type Culture Collection (ATCC, Manassas, USA), and cultured with Dulbecco's modified Eagle's medium (DMEM, Wisent, Nanjing, China) containing 10% (v/v) fetal bovine serum (FBS, Gibco, Gaithersburg, MD, USA) at 37°C with 5% CO₂. Fibroblasts were pretreated with serum-free medium for 12 h and then exposed to 5 ng/ml TGF-β1, 50 ng/ml IL-6, 200 ng/ml IL-6R (R&D Systems, MN, USA), or 500 ng/ml Fc-gp130 for 3, 6, or 12 h.

Chen W., Yuan H., Cao W., Wang T., Chen W., Yu H., Fu Y., Jiang B., Zhou H., Guo H, & Zhao X. (2019). Blocking interleukin-6 trans-signaling protects against renal fibrosis by suppressing STAT3 activation. Theranostics, 9(14), 3980-3991.

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Inflammatory Signaling in Endothelial Cells

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HUVECs (3 × 10⁵ cells/well for 6-well plates and 6 × 10⁴ cells/well for 24-well plates) were plated overnight in complete endothelial medium containing antibiotics. The next day, the medium was replaced with fresh antibiotics free medium and cells were treated with different concentrations of IL-6, IL-6R, TNF-α and LPS (all from R&D systems, USA) and/or pharmacological inhibitors CP690550, Stattic and LY294002 (all from R&D systems, USA), and PD98059 (Santa Cruz biotechnology, USA) for different time points ranging from 5 min to 48 h. At the end of incubations, supernatants and cells were collected and kept at -80 °C until further analysis. The cells were used to extract total protein or RNA.

Zegeye M.M., Lindkvist M., Fälker K., Kumawat A.K., Paramel G., Grenegård M., Sirsjö A, & Ljungberg L.U. (2018). Activation of the JAK/STAT3 and PI3K/AKT pathways are crucial for IL-6 trans-signaling-mediated pro-inflammatory response in human vascular endothelial cells. Cell Communication and Signaling : CCS, 16, 55.

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IL-6 Stimulation of FLSs

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Fresh ST samples were minced and digested in tissue enzyme digestion solution. FLSs were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS). The cells were seeded in 6-well plates (BD Biosciences, Bedford, MA, USA) at a density of 2 × 10⁵ cells/well and were maintained in a complete medium. After overnight serum starvation, the cells were stimulated with IL-6 (100 ng/mL; R&D Systems, Minneapolis, MN, USA) and IL-6 receptor (IL-6R; 100 ng/mL; R&D Systems).

Ikari Y., Isozaki T., Tsubokura Y, & Kasama T. (2019). Peficitinib Inhibits the Chemotactic Activity of Monocytes via Proinflammatory Cytokine Production in Rheumatoid Arthritis Fibroblast-Like Synoviocytes. Cells, 8(6), 561.

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Neutrophil Activation by OSCC Secretome

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Neutrophil were suspended in RPMI (10% FBS, 1% P/S) or conditioned media harvested from OSCC cell lines (CAL‐27, SCC‐4, SCC‐25) plated at 5 × 10⁶ mL⁻¹ in 96‐well culture plates (Corning) and then cultured for up to 40 h. Cytokine blocking experiments were conducted by culturing neutrophils in OSCC‐derived conditioned media, previously pre‐incubated for 30 min at 37°C in the presence of specific neutralising monoclonal antibodies (mAb) towards GM‐CSF (10 μg mL⁻¹, R&D Systems, Minneapolis, MN). Alternatively, neutrophils were pre‐incubated with IL‐6R (10 μg mL⁻¹ for 30 min, R&D Systems). After the incubation, neutrophils were then centrifuged at 300 × g for 5 min and finally suspended in 50 µL phosphatase‐buffered saline (PBS) at 1 × 10⁶ mL⁻¹ containing 2% FBS and 2 mm EDTA for flow cytometry assays.

Lonardi S., Missale F., Calza S., Bugatti M., Vescovi R., Debora B., Uppaluri R., Egloff A.M., Mattavelli D., Lombardi D., Benerini Gatta L., Marini O., Tamassia N., Gardiman E., Cassatella M.A., Scapini P., Nicolai P, & Vermi W. (2021). Tumor‐associated neutrophils (TANs) in human carcinoma‐draining lymph nodes: a novel TAN compartment. Clinical & Translational Immunology, 10(2), e1252.

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Immunofluorescence Analysis of Neural Markers

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Routine reagents were purchased from Sigma unless indicated otherwise. MBP84-104 (ENPVVHFFKNIVTPRTPPPSQ) and scrambled (s)MBP84-104 (NKPQTNVVEPFHRTFPIPPVS) peptides, derived from the human MBP sequence (GenBank #AAH08749), were synthesized by GenScript. The peptides were protected from degradation by exoproteinases using N-terminal acetylation and C-terminal amidation. The following primary antibodies were used in our immunofluorescence analyses: goat polyclonal IL-6 [R&D Systems (AF506), 1:100], goat polyclonal IL-6 receptor [IL-6R, R&D Systems (AF1830), 1:100], rabbit polyclonal glial fibrillary acidic protein [GFAP, DAKO (Z0334), 1:500], mouse monoclonal NeuN [EMD Millipore (MAB377), 1:1000], rabbit ionized Ca²⁺-binding adapter molecule 1 [Iba1, Wako (019-19741), 1:500], mouse neurofilament 200 [NF200, Millipore (MAB5262), 1:200], rabbit polyclonal calcitonin gene-related peptide [CGRP, Abcam (ab47027), 1:400], and rabbit polyclonal activating transcription factor 3 [ATF3, C-19 clone, Santa Cruz Biotechnology (SC-188), 1:100].

Ko J.S., Eddinger K.A., Angert M., Chernov A.V., Dolkas J., Strongin A.Y., Yaksh T.L, & Shubayev V.I. (2016). Spinal activity of interleukin 6 mediates myelin basic protein-induced allodynia. Brain, behavior, and immunity, 56, 378-389.

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Quantification of Inflammatory Mediators

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Cells were treated with vehicle (BSA+EtOH) and different concentrations of LA for 24 hours. Cell culture supernatants were collected and immediately stored at -80 °C. The concentrations of interleukin-6 (IL-6-R&D system, MN, USA), prostaglandin E 2 (PGE 2 -Cayman chemicals, MI, USA) and leukotriene B 4 (LTB 4 -Cayman chemicals) in cell culture supernatants were quantified by ELISA, according to the manufacture's protocols. For IL-6, optical density (OD) was measured at 450 nm in a microplate reader. A second reading at 540 nm was subtracted from the reading at 450 nm to correct for optical imperfections in the plate. PGE 2 and LTB 4 ELISAs were competitive assays and the absorbance was measured in microplate reader at a wavelength of 420 nm. The concentrations in cell supernatant were calculated from the provided standard curve.

Shrestha N., Cuffe J.S.M., Holland O.J., Perkins A.V., McAinch A.J, & Hryciw D.H. (2019). Linoleic Acid Increases Prostaglandin E2 Release and Reduces Mitochondrial Respiration and Cell Viability in Human Trophoblast-Like Cells. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 52(1).

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