OVA, LPS and Andrographolide were purchased from Sigma-Aldrich (St. Louis, MO). Water-soluble Andrographolide sulfonate (Xi-Yan-Ping Injection) was provided by Jiangxi Qingfeng Pharmaceutical Co., Ltd. ELISA kits for TNF-α, IL-6, IL-4 and IL-1β were purchased from Dakewei (Beijing, China). Anti-phosphorylation of p65 and anti-p-p65 were purchased from Cell Signaling Technology (Beverly, MA). Anti-NLRP3 and anti-CASP1 (3345-1) were purchased from Epitomics. Anti-ASC and anti-Actin (sc-1616) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-mouse CD3-FITC, CD11b-PE and CD11c-APC antibodies were bought from eBioscience. GTVisin™ anti-mouse/anti-rabbit immunohistochemical analysis KIT was purchased from Gene Company (Shanghai, China). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO).
Anti nlrp3
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Anti-NLRP3 is an antibody used for the detection and quantification of NLRP3 protein. NLRP3 is a member of the NLR family of proteins and plays a role in the formation of the inflammasome complex, which is involved in the regulation of inflammatory responses.
Automatically generated - may contain errors
Lab products found in correlation
Anti caspase 1, by Abcam (34 mentions)
Anti il 1β, by Abcam (20 mentions)
Anti asc, by Abcam (14 mentions)
Anti β actin, by Abcam (12 mentions)
Anti asc, by Santa Cruz Biotechnology (7 mentions)
Anti gsdmd, by Abcam (7 mentions)
Anti gapdh, by Abcam (7 mentions)
Anti caspase 1, by Santa Cruz Biotechnology (6 mentions)
Pvdf membrane, by Merck Group (6 mentions)
Anti β actin, by Cell Signaling Technology (5 mentions)
Anti β actin, by Proteintech (5 mentions)
Bca kit, by Beyotime (5 mentions)
Anti il 18, by Abcam (5 mentions)
Fluoview fv1000, by Olympus (5 mentions)
Anti gapdh, by Santa Cruz Biotechnology (4 mentions)
Anti txnip, by Abcam (4 mentions)
Anti zo 1, by Abcam (4 mentions)
Anti α sma, by Abcam (4 mentions)
Ripa lysis buffer, by Beyotime (4 mentions)
Anti pro caspase 1, by Abcam (4 mentions)
111 protocols using anti nlrp3
1
Andrographolide Modulates Inflammatory Pathways
2
Evaluation of MI-2 and Mepazine Compounds
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MI-2 and mepazine (chemical structure shown in Figure 1A , synthetic compounds provided by Eternity Bioscience Inc. NJ, USA) was dissolved at a concentration of 30 mM in 100% DMSO as a stock solution, stored at −20°C, and diluted with medium before each experiment. The final DMSO concentration did not exceed 0.1% throughout the study (all the control groups are composed of 0.1% DMSO). Cyclosporine A (CsA), phorbol myristate acetate (PMA), lipopolysaccharide (LPS) and adenosine triphosphate (ATP) were purchased from Sigma-Aldrich (St. Louis, MO). Dextran sulfate sodium (DSS, 36–50 kDa) was bought from MP Biomedicals (Aurora, OH). Myeloperoxidase (MPO) activity assay kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). RPMI-1640, FBS, Alexa Fluor 546 Donkey Anti-Rabbit IgG and Alexa Fluor® 488 Donkey Anti-Mouse IgG (H+L) were purchased from Life technology (Carlsbad, CA). Anti-phospho-IκBα, anti-phospho-IKKα/β, were purchased from Cell Signaling Technology (Beverly, MA). Anti-NLRP3, anti-phospho-p65 and anti-CASP1 were purchased from Epitomics (Burlingame, CA). Anti-ASC and anti-COX2 were purchased from Santa Cruz (Santa Cruz, CA). ELISA kits for murine TNF, IL-1β, IL-6, IFN-γ and human IL-1β were purchased from Dakewe Biotech Co. Ltd (Beijing, China). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).
3
Hyperuricemia Intervention Protocol
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The following materials and reagents were used in this study: geniposidic acid, verbascoside, typhaneoside, isorhamnetin-3-o-neohesperidoside, β-ecdysterone and chlorogenic acid chemical composition standard (all purity >95%) were obtained from Yuanye Biotechnology Co., Ltd. (Shanghai, China). Ethambutol (Ethb) (Jinhua, Chengdu, China). Adenine (AD) (Sigma-Aldrich, USA). Allopurinol (Allo) (Shimao Tianjie, Yancheng, China). UA, SCr, BUN test kits (Dibao Medical Supplies, Guangzhou, China). Urate staining solution (Gomori), hematoxylin eosin (HE) (Solarbio, Beijing, China). Interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-18 (IL-18) Enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, USA). The following primary antibodies were used for immunoblotting: anti-ABCG2 (Cat No. 4477s, 1:1,000, Cell Signaling Technology, USA), anti-NLRP3 (Cat No. ab263899, 1:1,000, Abcam, UK) and anti-GAPDH (Cat No. sc-365062, 1:1,500, Santa Cruz Biotechnology, USA).
4
Immunofluorescence Analysis of NLRP3 Inflammasome Components
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Paraffin-embedded sections of the ileum and gastric antrum were dewaxed, rehydrated, and subjected to antigen heat retrieval in citrate buffer followed by blocking with 5% bovine serum albumin (BSA) for 30 min at room temperature. Next, sections were incubated with rabbit anti-NLRP3 (dilution 1 : 1500; Abcam, Cambridge, UK), anti-ASC (1 : 1000; Abcam, Cambridge, UK), and anti-caspase-1 (dilution 1 : 500; Santa Cruz Biotechnology, CA, USA) primary antibodies overnight at 4°C. After washing with PBS 3 times, slides were stained with Cy3 conjugated goat anti-rabbit IgG, FITC conjugated goat anti-rabbit IgG, or FITC conjugated goat anti-mouse IgG (Wuhan Servicebio Technology Co., Ltd., Wuhan, China) secondary antibodies for 1 h at room temperature. Sections were washed three times, and nuclei were stained with 4′,6-diamidino-2- phenylindole (DAPI) for 10 min at room temperature. Images were captured using a fluorescence microscope (Eclipse C1, Nikon, Tokyo, Japan). For colocalization analysis, all sections were taken randomly and analyzed using ImageJ software, and the summarized colocalization efficiency data were represented by Pearson's correlation coefficient (PCC) [36 (link)]. The fluorescence intensity of caspase-1 in each group was also measured by ImageJ software.
5
NLRP3 Protein Expression Analysis
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GMSCs were lysed using RIPA buffer. The details of method for western blot were depicted as previously [24 (link)]. The expressions of protein were detected using anti-NLRP3 (1:300, Abcam), and β-actin (1:2000, Abcam) was used as an internal control. The secondary antibodies were obtained from the commercial companies: anti-mouse IgG (1:2000, Abcam) and anti-rabbit IgG (1:5000, Abcam).
6
Western Blot Analysis of NLRP3 Inflammasome
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After cell collection and total protein extraction, the proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and then transferred onto poly vinylidene fluoride membranes. The membranes were blocked with 5% skim milk at room temperature for 2 h, washed three times with tris-buffered saline Tween (TBS-T) solution and incubated with a primary antibody at 4 °C overnight. Subsequently, the membranes were incubated with anti-NLRP3 (1:1000 Abcam, Cambridge, MA, USA), ASC (1:1000 Affinity, America), caspase-1 (1:1000 Affinity, America), P-caspase-1 (1:1000 Abcam, MA, USA), iNOS and Arg-1 (1:1000 Zen BioScience, China) primary antibodies. The membranes were washed thrice with TBS-T and incubated with horseradish peroxidase-conjugated secondary antibodies (Sparkjade, China) for 2 h at room temperature. After subsequent washing with TBS-T solution thrice, protein bands were detected using an ECL (Affinity, America) kit. Finally, the band intensities were quantitatively analysed using ImageJ analysis software.
7
Protein Expression Analysis by Western Blot
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Protein expression was analyzed by Western blot. The total protein (50 μg) was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After the protein is transferred to the polyvinylidene fluoride microporous membrane (Bio-Rad), the membrane is blocked with 5% skimmed milk powder and the primary antibody [anti-Bax, anti-PCNA, anti–β-catenin, anti-Drp1 (1:1,000 dilution, American Abcam), anti-NLRP3 (1:500 dilution, American Abcam), anti-ASC, or anti–caspase-1]; then anti-mouse (anti-rabbit (IgG)) is linked with fluorescein (1: 1,000), and then incubated with an antibody conjugated to anti-fluorescein alkaline phosphatase (1:5,000). The immune complexes were detected with enhanced chemiluminescence reagents. For quantification, the signal optical density was normalized to β-actin by Quantity One image analysis software.
8
Quantifying Drp1 and NLRP3 in Colon Tissue
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The paraffin-embedded colon sections were deparaffinized, rehydrated, and pretreated with hydrogen peroxide in a PBS buffer. Heat-induced antigen retrieval was performed. The sections were incubated with anti-Drp1 (1:1,000 dilution, Abcam, United States) and anti-NLRP3 (1:100 dilution, Abcam, United States). The sections were incubated with HRP-conjugated secondary antibody, tyramide amplification was performed first, streptavidin-HRP was added, and then the DAB kit was used to show a positive signal. The sections were inspected at 400× magnification and analyzed using NIS-Elements. The following formula was used to calculate the positive content: positive content (PC) = average optical density x positive area.
9
NLRP3 Inflammasome Activation Pathway
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C2-Cer, LPS, adenosine 5′-triphosphate (ATP) disodium salt hydrate, SN50, verapamil, imipramine, sulfo-N-succinimidyl oleate (SSO) and phorbol 12-myristate 13-acetate (PMA) were acquired from Sigma-Aldrich (St Louis, MO, USA). Anti-NLRP3, anti-TXNIP, anti-CD36, anti-nuclear factor-κB (NF-κB) and anti-p-NF-κB antibodies were purchased from Abcam (San Francisco, CA, USA). The secondary antibody was obtained from ZSGB-BIO (Beijing, China). Anti-caspase-1, anti-β-actin, and lysis buffer were obtained from Cell Signaling Technology (Beverly, MA, USA).
10
Immunofluorescence Staining of ARPE-19 Cells
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After plating the ARPE-19 cells onto six-well plates and generating cells grown on a coverslip, the coverslip was washed once with a PBS buffer, and then the cells would be fixed for 20 min at room temperature. To break the membrane, 0.2% Triton X-100 was added following with three times washes with PBS. Incubation with blocking solution (PBS containing 10% goat serum) was conducted for 2 h at 4°C and then the cells would be incubated with dilute primary antibody (anti-ZO1, Cat No.: ab221547, Abcam, UK; anti-NLRP3, Cat No.: ab263899, Abcam, UK) for immunostaining in a cold room overnight. The cells were washed three times with PBS and then the secondary antibody was added to incubate with the cells for 1 h in the dark. Drop the DAPI & anti-quencher mixture onto the glass slide and flip the cover glass. Observation and record of the immunofluorescence photographs were conducted with a Zeiss confocal microscope (Leica Biosystems, Germany) under dark.
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