Il 1ra

Manufactured by Abcam

Sourced in United States

IL-1RA is a protein that functions as an antagonist to the pro-inflammatory cytokine Interleukin-1 (IL-1). It binds to the IL-1 receptor, thereby preventing the binding and activation of IL-1.

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Anti-IL-1Ra (4 citations) Rabbit anti-IL-1RA (3 citations) Human IL-1RA (2 citations)

7 protocols using il 1ra

Immunoblotting of Inflammatory Markers

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The cell lysates were prepared as described previously
[24 (link)]. Western blot analysis was performed using primary antibodies against inducible nitric oxide synthase (iNOS) (0.1 μg/ml), TNFα (0.1 μg/ml), IL-1β (0.1 μg/ml), TGFβ (0.125 μg/ml), CD14 (0.1 μg/ml), soluble tumor necrosis factor receptor (sTNFR) type II (2 μg/ml), IL-1RA (2.5 μg/ml) (Abcam, Cambridge, MA, USA), or CD68 (0.9 μg/ml; Epitomics, Burlingame, CA, USA). The density of protein bands was assessed using a computing densitometer with Image-Pro plus software (Media Cybernetics, Inc., Bethesda, MD, USA).

Lien G.S., Liu J.F., Chien M.H., Hsu W.T., Chang T.H., Ku C.C., Ji A.T., Tan P., Hsieh T.L., Lee L.M, & Ho J.H. (2014). The ability to suppress macrophage-mediated inflammation in orbital fat stem cells is controlled by miR-671-5p. Stem Cell Research & Therapy, 5(4), 97.

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Cytokine Profiling in LOMM and 3D-BSGM Models

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The levels of cytokines (IL-1RA, IL-10, CCL2, IFNα, IFNγ, GM-CSF, and TNFα; purchased from Abcam, Cambridge, USA) were measured and compared in media collected from the LOMM models. For mature IL-1β, we used the IL-1 beta Human ELISA Kit (Thermo Fisher Scientific, USA, Cat# BMS224-2). We also measure the cytokine level in media collected from the 3D-BSGM models per the manufacturers' instructions.

Shahraki A.H., Tian R., Zhang C., Fregien N.L., Bejarano P, & Mirsaeidi M. (2022). Anti-inflammatory Properties of the Alpha-Melanocyte-Stimulating Hormone in Models of Granulomatous Inflammation. Lung, 200(4), 463-472.

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Multimodal Immunohistochemical Characterization

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Cells or spinal cord sections were fixed with 4% paraformaldehyde for 15 min, permeabilized in 0.2% Triton X-100 for 30 min, then blocked in 10% BSA for 1 h and finally incubated overnight at 4°C with primary antibodies against the following proteins: GFAP (1:1000, Abcam), NeuN (1:500, Abcam), OX42 (1:200, Serotec), MAP2 (1:500, Abcam), CD11b (1:200, Abcam), CCL2 (1:250, Serotec), CCR2 (1:50, Santa Cruz), and IL-1Ra (1:100, Abcam). After incubation, cells or slides were rinsed gently in PBS three times and then incubated with Alexa Fluor 488- and Alexa Fluor 594-conjugated secondary antibodies (1:200, Jackson ImmunoResearch, USA) for 2 h at room temperature. Finally, nuclei were counterstained with DAPI. Images were captured under a fluorescence microscope with equal exposure times.

Rong Y., Ji C., Wang Z., Ge X., Wang J., Ye W., Tang P., Jiang D., Fan J., Yin G., Liu W, & Cai W. (2021). Small extracellular vesicles encapsulating CCL2 from activated astrocytes induce microglial activation and neuronal apoptosis after traumatic spinal cord injury. Journal of Neuroinflammation, 18, 196.

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Immunofluorescence Assay for Apoptotic Markers

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Immunofluorescence analysis was performed, as previously described^{16 ,17 (link)}. Briefly, NP cells or tissues attached to slides were fixed with 4% paraformaldehyde and then permeabilized with 0.2% Triton X-100 (Beyotime Biotechnology, China) in PBS. The slides were washed in PBS, blocked with 2% bovine serum albumin in PBS for 2 h at 37 °C and then incubated with primary antibodies against caspase-3 (1:100; Cell Signaling Technology, Danvers, MA), caspase-9 (1:100; Cell Signaling Technology, Danvers, MA), IL-1β (1:100; Abcam, Cambridge, UK), IL-1Ra (1:100; Abcam, Cambridge, UK), COL2A1 (1:100; Abcam, Cambridge, UK), and FAS (1:150; Proteintech Group, China). After being washed twice, the slides were subsequently treated with a secondary goat anti-rabbit antibody (Boster Biological Technology, China) at 37 °C for 2 h. Nuclei were costained for 5 min with 0.1 g/mL 4-6-diamidino-2-phenylindole (Beyotime Biotechnology, China). Images were captured under a microscope (Olympus, BX53; Melville, NY, USA).

Wu X., Liao Z., Wang K., Hua W., Liu X., Song Y., Zhang Y., Yang S, & Yang C. (2019). Targeting the IL-1β/IL-1Ra pathways for the aggregation of human islet amyloid polypeptide in an ex vivo organ culture system of the intervertebral disc. Experimental & Molecular Medicine, 51(9), 1-16.

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Western Blot Analysis of Inflammatory Markers

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Proteins were extracted from cells and tissues, and protein concentrations were measured with BCA assays. Equal amounts of protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. Membranes were blocked with 5% bovine serum albumin for 1 h at room temperature and then incubated overnight at 4 °C with antibodies against CCR2 (1:1000, Serotec), Cleaved caspase-3 (1:1000, Cell Signal Technology), Bcl-2 (1:1000, Abcam), Bax (1:1000, Abcam), TNF-α (1:1000, Abcam), IL-1β (1:1000, Abcam), IL-6 (1:1000, Abcam), IL-1Ra (1:1000, Abcam), OX42 (1:1000, Serotec), and GAPDH (1:1000, Abcam). After the membranes were washed with Tris-buffered saline with Tween, they were incubated for 2 h at room temperature with HRP-conjugated secondary antibodies (1:2000, Thermo Fisher Scientific, USA). The bands were visualized with enhanced chemiluminescence reagent (Thermo Fisher Scientific, USA), and the quantification of target proteins was performed in ImageJ (National Institutes of Health, Bethesda, MD, USA).

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Intrathecal Administration of Pharmacological Agents

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Remifentanil hydrochloride was purchased from Guangzhou Renfu Medical Equipment Co., Ltd, dissolved in saline (NaCl 0.9%), and infused intravenously at a rate of 1.2 μg/kg/min for 60 min, at our previously reported dose.^{7 (link)} Controls received the same volume of saline under identical conditions. Sevoflurane was purchased from Maruishi Pharmaceutical Co.; I IL-1β inhibitor antagonist (IL-1ra) from Abcam, A438079 from Tocris, and (+)-Naloxone, ac-YVAD-cmk, and DMSO from Merck KGaA. For intrathecal drug delivery, the catheters were preloaded with drugs at the distal end in a total volume and delivered over 20–30 s from when the catheter was in position. Intrathecal doses were as follows: IL-1ra, 100 μg in 10 μL; (+)-Naloxone, 1,200 μg in 10 μL; A438079, 600 ng in 10 μL; and ac-YVADcmk, 20 μg in 10 μL, based on the dose reported by Grace, et al.^{10 (link),12 (link)} A total of 0.15 μl DMSO dissolved in 10 μl saline was used as a vehicle in each group.

Yuan Y., Zhao Y., Shen M., Wang C., Dong B., Xie K., Yu Y, & Yu Y. (2022). Spinal NLRP3 inflammasome activation mediates IL-1β release and contributes to remifentanil-induced postoperative hyperalgesia by regulating NMDA receptor NR1 subunit phosphorylation and GLT-1 expression in rats. Molecular Pain, 18, 17448069221093016.

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Protein Expression Analysis in Liver Tissues

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Western blotting was performed to analyze the expression of NLRP3 (ABclonal, Woburn, MA, USA), caspase-1 (casp-1; Sigma-Aldrich), IL-1β (Abcam, Cambridge, UK), IL-18 (R&D Systems, Minneapolis, MN, USA), IL-1RA (Abcam), and β-actin (Cell Signaling Technology, Danvers, MA, USA) in liver tissues according to the manufacturer’s protocol. Serum concentrations of TGF-β (R&D Systems), IL-6 (R&D systems), IL-10 (Thermo Fisher Scientific, Waltham, MA, USA), and IL-18 (Thermo Fisher Scientific) were detected using an ELISA kit according to the manufacturer’s protocol.

Chen K., Obara H., Matsubara Y., Fukuda K., Yagi H., Ono-Uruga Y., Matsubara K, & Kitagawa Y. (2022). Adipose-Derived Mesenchymal Stromal/Stem Cell Line Prevents Hepatic Ischemia/Reperfusion Injury in Rats by Inhibiting Inflammasome Activation. Cell Transplantation, 31, 09636897221089629.

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