Lipopolysaccharide (LPS) and type I collagenase were purchased from Sigma-Aldrich Co. Ltd. (Sigma, MO, USA). Trizol, Dulbecco's modified Eagle culture medium (DMEM), penicillin-streptomycin mixture, and fetal bovine serum (FBS) were purchased from Gibco (Rockville, USA). The primary antibodies of β-actin, NGF, TrKA, TRPV1, IL-1β, and PGP 9.5 were purchased from Bioss (Beijing China). The Sirius red staining kit and goat anti-rabbit IgG H&L (HRP) kit were also purchased from Abcam (Cambridge, UK). 5 × HiScript II qRT SuperMix and 2 × Chamq SYBR qPCR MasterMix were purchased from Vazyme (Nanjing, China). Primers were supplied by Generay Biotechnology (Shanghai, China). ELISA kits for NGF and SP were purchased from JinyiBai Company (Nanjing, China).
Il 1β
Manufactured by Bioss Antibodies
Sourced in China, United States
IL-1β is a cytokine that plays a crucial role in the regulation of immune and inflammatory responses. It is an important mediator of the body's response to injury, infection, and other forms of inflammation.
Automatically generated - may contain errors
Lab products found in correlation
Interleukin 6 (il 6), by Bioss Antibodies (6 mentions)
Tnf α, by Bioss Antibodies (5 mentions)
Ripa lysis buffer, by Beyotime (4 mentions)
β actin, by Bioss Antibodies (3 mentions)
Gapdh, by Proteintech (2 mentions)
Ifn γ, by Bioss Antibodies (2 mentions)
Iba 1, by Abcam (2 mentions)
Interleukin 6 (il 6), by Abcam (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Pgp 9, by Bioss Antibodies (1 mentions)
Goat anti rabbit igg h l hrp kit, by Abcam (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Collagenase type 1, by Merck Group (1 mentions)
Dulbecco s modified eagle culture medium dmem, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin mixture, by Thermo Fisher Scientific (1 mentions)
Trpv1, by Bioss Antibodies (1 mentions)
Sirius red staining kit, by Abcam (1 mentions)
Hiscript 2 q rt supermix, by Vazyme (1 mentions)
Chamq sybr qpcr master mix, by Vazyme (1 mentions)
Lipopolysaccharide lps, by Merck Group (1 mentions)
21 protocols using il 1β
1
LPS and Collagenase Induced Inflammation
2
Cardiac Protein Profiling via Western Blot
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Fifty milligrams of myocardial tissue was collected, and total protein was extracted from tissue lysates. The protein concentration was determined using the BCA method. Appropriate protein samples were separated using SDS-PAGE electrophoresis, and the proteins were transferred to PVDF membranes via electrical imprinting transfer. Antibodies against cleaved caspase-3, Bax, Bcl-2, light chain 3B (LC3B), Beclin-1 (1 : 1000, Cell Signaling Technology, USA), PK2 (1 : 1000, Abcam, USA), PKR1, PKR2 (1 : 2000, Santa Cruz Biotechnology, USA), GAPDH (1 : 10000, Proteintech, USA), ubiquitin-binding protein (p62) (1 : 500, Wanleibio, China), autophagy-related proteins (Atg5), NALP3, caspase-1, IL-18, and IL-1β (1 : 1000, Bioss, China) were incubated overnight at 4°C. Secondary antibodies were added and incubated at room temperature for 1 h. After ECL color development, Image Lab software was used to determine the band absorbance value. GAPDH expression was used as the loading control.
3
Measuring Inflammatory Markers in Rat Plasma
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Abdominal aortic blood was extracted from rats and placed in an anticoagulation tube for 2 h at room temperature and then centrifuged at 3000 r/min speed. The supernatant was absorbed with pipettes (Eppendorf, Hamburg, Germany) and stored at −80°C for testing. Plasma ET-1, interleukin 1β(IL-1β), interleukin 6 (IL-6), tumor necrosis factor α (TNF-α) (Bioss, Beijing, China), and cyclooxygenase 1 (COX-1) (Cusabio, Wuhan, China) levels of rats were detected according to ELISA kit instructions.
4
Annexin V-FITC/PI Apoptosis Assay
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An Annexin V-FITC/PI Apoptosis kit was obtained from BD Company (Franklin Lakes, USA). Modified Bradford Protein Assay Kit, Antibody against NLRP3, Animal Total RNA Isolation Kit and Tissue Total Protein Extraction Kit were supplied by Sagon Biotech Company (Shanghai, China). Another antibody against Gasdermin D (GSDMD) was bought from Thermo Fisher Company (USA). IL-1β, IL-18 and β-actin antibodies were obtained from Bioss Company (Beijing, China). Caspase-1 antibody was obtained from Boster Biological Technology com.Ltd (Wuhan, Hubei, China). A PrimeScript RT reagent kit was bought from TAKARA company (Daliang, China). PRV-HLJ strain (MK080279.1) is a strain isolated from Heilong Jiang prpvince and provided by Professor Jingfei Wang from Harbin Veterinary Research Institute, CAAS. Eighty-six-week-old female Balb/C mice were obtained from Dossy Experimental Animal Corporation (Chengdu, China).
5
Liver Protein Expression Analysis
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The liver tissues were homogenized with lysis buffer containing a protease inhibitor cocktail. The homogenates were centrifuged at 12,000 rpm for 20 min at 4°C. After the supernatant was collected, the protein concentration was determined using the Bio-Rad protein assay kit (Bio-Rad, Irvine, CA, USA). An equal amount of proteins were resolved on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, proteins were transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Burlington, MA, USA). The membrane was incubated with 5% skim milk for 50 min at room temperature and then washed with PBS-T. The membrane was incubated with a primary antibody overnight at 4°C and then washed with PBS-T. The primary antibodies used were: β-actin (1:1,000; Cell Signaling, Beverly, MA, USA), NF-κB p65 (1:500, Abcam, Cambridge, UK), iNOS (1:500, Merk, Kenilworth, NJ, USA), COX-2 (1:1,000, Cell Signaling, Beverly, MA, USA), IL-1β (1:500; Bioss Antibodies, Boston, MA, USA), SOD (1:500; Santa Cruz, Dallas, TX, USA), catalase (1:500; Santa Cruz), and GSH-Px (1:500, Santa Cruz). The membranes were incubated with secondary antibodies for 1 h. The immuno-complexes were visualized by enhanced chemiluminescence solution (ELPIS Biotech, Daejeon, Korea) and bands were visualized with a chemiluminescent imaging system (Davinci Chemi, Seoul, Korea).
6
Immunohistochemical Analysis of Inflammatory Markers
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The sections were stained with the following primary antibodies for the IHC analyses: IL-1β, IL-6, RANKL, and Cathepsin K (Bioss, Woburn, MA, USA) (1:400 dilution). After deparaffinization and rehydration, sections were treated with 3% hydrogen peroxide (Abcam) for 10 min to quench the endogenous peroxidase activity, followed by the incubation with normal goat serum to block the non-specific binding for 30 min at room temperature. Primary antibodies with different specific concentrations were applied to the sections and incubated overnight at 4 °C. The following day, the slides were incubated with the biotinylated secondary antibody for 30 min using the VECTASTAIN Elite ABC Rabbit IgG Kit (Vector Labs, Burlingame, CA, USA). Subsequently, the prepared VECTASTAIN ABC reagent was applied to the slides and incubated for another 30 min. Sections were stained with 3,3’-Diaminobenzidine (DAB) (Abcam) and counterstained with hematoxylin.
7
Western Blot Analysis of Inflammatory Proteins
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Proteins were extracted from RAW264.7 cells using radioimmunoprecipitation (RIPA) buffer (Yazyme, Shanghai). Protein concentration was determined by BCA protein detection kit (Biyuntian, Shanghai). SDS-PAGE was used to separate proteins from different samples. After transferring the separated proteins to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, CA, USA), the PVDF membranes were blocked with protein-free fast blocking solution (Yazyme, Shanghai) for 15 min. Blocked PVDF membranes were then incubated in diluted primary antibody for 12 h at 4°C. The dilution ratios of different antibodies (Boaosen, Beijing) were as follows: β-actin (1 : 1000), NF-κB-p65 (1 : 1000, Bioss), p-NF-κB-p65 (1 : 1000, Bioss), IL-1β (1 : 500, Bioss), IL-6 (1 : 500, Bioss), and TNF-α (1 : 500, Bioss). Next, the PVDF membrane and diluted secondary antibody were incubated for 1 h at room temperature. Finally, proteins on PVDF membranes were imaged using enhanced chemiluminescence (ECL) solution (GlpBio, USA), and protein bands were processed using ImageJ to detect protein expression levels.
8
Western Blot Analysis of Inflammatory Markers
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Primary antibodies against eNOS (1:2000), p‐eNOS (Ser1177) (1:1000), iNOS (1:2000), p65 (1:2000), Phospho‐p65 (Ser536) (1:1000), IκBα (1:2000), Phospho‐IκBα (Ser32) (1:1000), AMPKα (1:2000), Phospho‐AMPKα (Thr172) (1:1000), SIRT1 (1:2000) and SIRT3 (1:2000) were purchased from Cell Signaling Technology. Primary antibodies against NLRP3 (1:1000), Caspase‐1 (1:1000), ASC (1:1000), IL‐1β (1:1000), and 3‐nitrotyrosine (3‐NT) (1:1000) were obtained from Bioss (Beijing, China). Primary antibodies against TLR4 (1:2000), SOD2 (1:2000), NOX2 (1:2000), NOX4 (1:2000), GAPDH (1:5000) and secondary antibodies (goat‐anti‐rabbit) (1:5000) were obtained from Proteintech Group. Unless otherwise indicated, all other chemicals used in this study were obtained from Sigma‐Aldrich.
9
Chondrocyte and Synoviocyte Immunostaining
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The chondrocytes and synoviocytes were cultured with different treatments for 48 h.
The cell immuno-staining was performed according to the previous protocols 17 . The primary antibodies: Collagen II (Santa Cruz, sc-7763), MMP-13 (Santa Cruz, sc-30073) for chondrocytes, and IL-1β (Bioss, bs-20449R), MMP-13 (Santa Cruz, sc-30073) for synoviocytes were used. After different secondary antibodies incubation, the samples were observed and captured by fluorescent or bright field microscopy (Olympus).
The cell immuno-staining was performed according to the previous protocols 17 . The primary antibodies: Collagen II (Santa Cruz, sc-7763), MMP-13 (Santa Cruz, sc-30073) for chondrocytes, and IL-1β (Bioss, bs-20449R), MMP-13 (Santa Cruz, sc-30073) for synoviocytes were used. After different secondary antibodies incubation, the samples were observed and captured by fluorescent or bright field microscopy (Olympus).
10
Molecular Mechanisms of Anti-Inflammatory Action
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Sodium taurocholate and emodin were purchased from Aladdin Regents (Shanghai, China). ML385 was purchased from MedChemExpress (New Jersey, USA). Dexamethasone (DEX) was purchased from Yucheng Kelong Veterinary Medicine (Shanxi, China). NLRP3 and ASC antibodies were purchased from ABclonal (Wuhan, China). IL-18, cleaved-caspase 1, IκBα, p65, Nrf2 and HO-1 antibodies were purchased from Proteintech (Illinois, USA). IL-1β and p-IκBα antibodies were purchased from Bioss (Beijing, China). p-p65 antibody was purchased from MultiSciences Biotech (Hangzhou, China). Histone H3 antibody was purchased from ABGENT (California, USA). β-actin antibody was purchased from Santa Cruz Biotechnology (California, USA). Goat anti-Rabbit IgG and Goat anti-Mouse IgG were purchased from Beyotime Biotechnology (Shanghai, China).
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