ODZ was provided by Professor Sang-Kyu Ye (Department of Pharmacology, Seoul National University College of Medicine, Seoul, Republic of Korea) [21 (link)]. Penicillin-streptomycin, trypsin-EDTA, Fetal bovine serum, RPMI 1640, DMEM-high glucose pyruvate, and Opti-MEM were purchased from Gibco (Grand Island, NY, USA). LPS (O111:B4), ATP and DMSO were purchased from Sigma-Aldrich (St. Louis, MO, USA). Nigericin, nano-SiO2, imiquimod, poly(dA:dT), MSU crystal, Y-VAD-CMK, MCC950, lipofectamine-2000, and mouse IL-1β ELISA kit were purchased from InvitroGen (San Diego, CA, USA). Flagellin was purchased from Adipogen (San Diego, CA, USA). Mouse IL-1β antibody (AF-401-NA) and human IL-1β ELISA kit were purchased from R&D Systems (Minneapolis, MN, USA). Antibodies against mouse caspase-1 (AG-20B-0042-C100), ASC (AG-25B-0006-C100), and NLRP3 (AG-20b-0014-C100) were purchased from Adipogen (San Diego, CA, USA), and mGSDMD (ab209845) was purchased from Abcam (Cambridge, UK). Anti-FLAG antibody (F1804) and β-Actin antibody (SC-4778) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Santa Cruz biotechnology (Dallas, TX, USA), respectively.
Ag 20b 0014 c100
Manufactured by Adipogen
Sourced in United States
AG-20B-0014-C100 is a laboratory equipment product. It serves a core function without any interpretation or extrapolation on its intended use.
Automatically generated - may contain errors
Lab products found in correlation
Ag 20b 0042 c100, by Adipogen (6 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
Ag 25b 0006, by Adipogen (2 mentions)
Sa00001 1, by Proteintech (2 mentions)
Sa00001 2, by Proteintech (2 mentions)
Ve cadherin, by Santa Cruz Biotechnology (2 mentions)
Af 401 na, by Santa Cruz Biotechnology (2 mentions)
Caspase 4, by Santa Cruz Biotechnology (2 mentions)
Ag 20b 0042 c100, by Novus Biologicals (2 mentions)
Caspase 5, by Santa Cruz Biotechnology (2 mentions)
Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (2 mentions)
Cytotox 96 non radioactive cytotoxicity assay kit, by Promega (2 mentions)
β actin, by Merck Group (2 mentions)
Polyclonal anti sars cov 2 spike glycoprotein, by BEI Resources (2 mentions)
O dianisidine dyhydrochloride, by Merck Group (2 mentions)
Af 401 na, by R&D Systems (2 mentions)
Ab241996, by Abcam (2 mentions)
Ag 20b 0042, by Adipogen (2 mentions)
Sc 271054, by Santa Cruz Biotechnology (2 mentions)
Lsm 510 meta confocal microscope, by Zeiss (2 mentions)
36 protocols using ag 20b 0014 c100
1
Inflammasome Activation Pathway Protocol
2
Protein Extraction and Western Blot Analysis
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Supernatants were collected and cell pellets were resuspended in lysis buffer containing protease inhibitor cocktail (Roche, Basel, Switzerland). Cell lysates were stored at–20 °C o/n, then centrifuged at 13000 rpm for 10 min. Protein concentration was measured by Bradford assay. Supernatants were precipitated by adding an equal volume of methanol and 1:4 volume chloroform followed by 10 min centrifugation at 12000 rpm, RT. After removal of the upper layer, two-volumes methanol was added and samples were centrifuged again. The liquid phase was discarded, the pellet dried at 50 °C for 5 min then dissolved in reducing sample buffer, heated at 90 °C for 5 min then subjected to electrophoresis using 4–12% Criterion™ XT Bis-Tris Gels (Bio-Rad Laboratories) with MES buffer. For western blot analysis the following antibodies were used: antibody against IL-1β (AF-201-NA) was purchased from R&D Systems; antibody against caspase-1 (sc-515) was from Santa Cruz Biotechnology; antibody against NLRP3 (AG-20B-0014-C100) was from Adipogen. All secondary antibodies were purchased from Santa Cruz Biotechnology.
3
Immunohistochemical Staining of Inflammatory Markers
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Following deparaffinization and rehydration, sections were treated with a 10-mM citrate buffer (Cat No.AP-9003-125 Labvision). Endogenous peroxidase was inactivated in 3% H2O2, then incubated with normal serum blocking solution. Sections were incubated over 18 h at +4 °C overnight with AIF-1(allograft inflammatory factor 1, AB-70353, Elabscience, Texas, USA), NLRP3 (AG-20B-0014-C100, Adipogen, San Diego, CA, USA), and IL1β (SC7884, Santa Cruz, Darmstadt, Germany) primer antibodies. After washing, sections were then incubated with biotinylated IgG and then with streptavidin conjugated to horseradish peroxidase (Invitrogen 85–9043). Sections were stained with DAB and counter-stained with Mayer’s hematoxylin and analyzed using a light microscope [17 (link)].
4
Western Blot Analysis of Inflammasome Proteins
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Cell lysates and culture supernatants were incubated with cell lysis buffer (20 mM Tris HCl pH 7.4, 200 mM NaCl, 1% NP-40) and denatured in laemmli buffer. Subsequently the protein samples were boiled at 95°C for 10 min and separated by SDS-PAGE. Separated proteins were transferred to PVDF membranes. Blocking, incubation with antibody and washing of the membrane were done in PBS supplemented with 0.05% Tween-20 (v/v) and 3% (w/v) non-fat dry milk. Immunoblots were incubated overnight with primary antibodies against caspase-1 (AG-20B-0042-C100, Adipogen), Nlrp3 (AG-20B-0014-C100, Adipogen), ASC (AG-25B-0006, Adipogen), IL-1β (GTX74034, Genetex), IL-18 (5180R-100, Biovision), IkBα (9242S, Cell Signaling), Phospho-IkBα (2859S, Ser32) (Cell Signaling), β-actin (NB600-501H, Novus Biologicals) and A20 (sc-166692, Santa Cruz Biotechnology). Horseradish peroxidase-conjugated goat anti-mouse (115-035-146, Jackson Immunoresearch Laboratories) or anti-rabbit secondary antibody (111-035-144, Jackson Immunoresearch Laboratories) was used to detect proteins by enhanced chemiluminescence (Thermo Scientific).
5
Protein Expression Analysis in THP-1 Cells
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Total protein was extracted from THP-1 cells using protein lysis solution. After protein quantification, electrophoresis and transmembrane, the protein samples were incubated with primary antibodies against vinculin (1:10,000 dilution; ab129002; Abcam, Cambridge, UK), AKT (1:1,000 dilution; 9271; CST, Danvers, MA, USA), phospho-AKT (1:1,000 dilution; 2211; CST), IL-1β (1:1,000 dilution; 12242; CST), IL-18 (1:1,000 dilution; ab243091; Abcam), nucleotide‐binding oligomerization domain (NOD)‐like receptor protein 3 (NLRP3; 1:1,000 dilution; AG-20B-0014-C100; AdipoGen, San Diego, CA, USA), caspase‐1 (1:10,000 dilution; ab108362; Abcam), NF‐κB p65 (1:1,000 dilution; AB32360; Abcam), or phospho‐NF‐κB p65 (1:1,000 dilution; 3033; CST) overnight at 4°C. Secondary antibodies conjugated with horseradish peroxidase (1:5,000 dilution; SA00001-1; SA00001-2; Proteintech, Rosemont, PA, USA) were incubated at room temperature for 1 h. The protein bands were visualized using an enhanced chemiluminescence kit (ECL; Advansta, Menlo Park, CA, USA). Data acquisition and analysis were performed on a gel image analyzing system equipped with Image Lab Software (Bio‐Rad, Hercules, CA, USA).
6
NLRP3 Inflammasome Protein Expression
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The collected THP-1 cells were lysed in a RIPA buffer. The expression of NLRP3 inflammasome was detected using immunoblotting with anti-NLRP3 (AG-20B-0014-C100, AdipoGen).
7
Co-immunoprecipitation of ER Proteins
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Co-immunoprecipitation was carried out using protein Gibco CTS dynabeads (Thermo Fisher) in accordance with the manufacturer’s instructions. Protein extractions from the ER fractions were carried out by adding 50 mM NaCl and 1% NP-40 to the homogenization buffer. All the buffers were supplemented with a proteases inhibitor mixture and Phos‐STOP Phosphatase Inhibitor Cocktail (Roche Applied Science). The same amount of extracted proteins for each condition was incubated O/N with the specific primary antibody PML (Millipore, no. MAB3738), P2X7 (Alomone, APR-004), or NLRP3 (AG-20B-0014-C100, Adipogen). The immunocomplexes were captured with the appropriate dynabeads (Thermo Fisher). Beads were pelleted and washed three times. The bait was eluted in Laemmli sample buffer and denatured for 5 min at 100 °C. Samples were proceeded by SDS–PAGE and analyzed by standard (WB) blotting technique.
8
Immunofluorescence Analysis of Neural Progenitor Cells
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Cultures were fixed in freshly prepared, buffered 4% paraformaldehyde. After blocking with 20% normal goat serum and permeabilization for 10min with 0.2% Triton X-100 in PBS, cultures were incubated overnight at 4°C with the following antibodies (mAb, monoclonal; pAb, polyclonal): Nestin ( MAB5326, Millipore, 1:200), Ki67 (ab16667, Abcam, 1:1000), Oct4 (MAB4401, Millipore, 1:2000), Pax6 (Developmental Studies Hybridoma Bank; 1:30), β-tubulin isotype III (β-tubIII, mAb, MMS-435P Covance, 1:400), Glial Fibrillary Acidic Protein (GFAP, z033429-2, Dako, 1:400), Lamp1 (pAb, ab24170, Abcam, 1:750), Cleaved Caspase-3 (pAb, #9661, Cell Signaling, 1:500), NLRP3 (pAb, AG-20B-0014-C100, AdipoGen, 1:200), Microtubule-Associated Protein 2 (MAP2, AB5622, Millipore, 1:400), Caspase 1 (Casp1, SC-515, Santa Cruz Biotechnology, 1:100), LC3B (pAb#2775, Cell Signaling, 1:400), GAPDH (ab9485, Abcam, 1:3000). After removal of the primary antibodies and repeated washes with PBS, cultures were incubated at room temperature for 45 min with secondary antibodies labeled with Alexa Fluor 633 or 488 (anti-mouse and/or anti-rabbit, Molecular Probes). Samples were then labeled with DAPI (0.3 μg/ml, Roche) for nuclear staining and rinsed with PBS for mounting and analysis. Microphotographs were taken using a confocal microscope.
9
Characterizing NLRP3 Inflammasome Pathway
10
Immunoblotting Analysis of Bone Marrow Cells
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Bone marrow adherent cells were cultured as previously described. All cells were lysed in RIPA buffer (Sigma, St. Louis, MS, USA) supplemented with protease inhibitors (Roche, Indianapolis, IN, USA), centrifuged at 13,000× g for 20 min at 4 °C, and the supernatants were obtained. Protein concentration was determined in the clarified supernatant using Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Proteins were electrophoresed on Mini-PROTEAN TGX 4−20% gels (Bio-Rad Laboratories) and transferred to nitrocellulose 0.2 mm (GE Water & Process Technologies, Feasterville-Trevose, PA, USA). Membranes were blocked in 5% rehydrated non-fat milk in PBS-Tween 0.5% for 1 h at room temperature. Primary antibodies for PD-L1 (1:250) (Catalog#AF1019) (R&D Systems), NLRP3 (1:1000) (Catalog#AG-20B-0014-C100) (Adipogen, San Diego, CA, USA), and IL-1β (1:250) (Catalog#AF-401-NA) (R&D Systems) were used in combination with peroxidase-conjugated secondary antibodies. A primary antibody against β-actin (Santa Cruz Biotechnology, Dallas, TX, USA) was used to assess protein loading. Chemiluminescence was captured using Bio-Rad Chemidoc MP Imaging System (Bio-Rad Laboratories). Images were quantified using ImageJ (U.S. National Institutes of Health, Bethesda, MD, USA).
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