BMDMs were isolated from bone marrow of C57BL/6 wild-type or Caspase-1−/− (Jackson Laboratory) female mice (7–8 weeks old) and differentiated for 7 days in RPMI 1640 medium supplemented with 10% fetal bovine serum (Gibco), penicillin (100 U/ml) and streptomycin (0.1 mg/ml) and 20 ng/ml human M-CSF (R&D Systems). THP-1 (Cell Bank of Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences) were cultured in RPMI 1640 medium with FBS and penicillin and streptomycin. Phorbol 12-myristate 13-acetate (100 ng/ml, 24 h) was used in differentiation of THP-1 into macrophages. The additives or inhibitors used in in vitro experiments are as follows: purified SAK (0.5 μg/ml), lipopolysaccharide (LPS, 0.2 μg/ml), ATP (5 mM), MCC950 (1 μM, Selleck), potassium channel blocker glibenclamide49 (link) (10 μM, Sigma), KCl (25 mM), NADPH oxidase inhibitor apocynin50 (link) (200 μM, Selleck), lysosome membrane stabilizer dexamethasone51 (link) (200 nM, Sangon Biotech), ROS scavenger NAC52 (link) (1 mM, Sigma), and NF-κB inhibitor BAY 11-7082 (10 μM, Sigma).
Apocynin
Manufactured by Selleck Chemicals
Sourced in United States
Apocynin is a chemical compound commonly used in research laboratories. It functions as an inhibitor, specifically targeting the enzyme NADPH oxidase. This enzyme plays a role in the production of reactive oxygen species. Apocynin is utilized in various scientific investigations to explore its effects on cellular processes and signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
N acetyl l cysteine nac, by Merck Group (2 mentions)
Streptomycin, by R&D Systems (1 mentions)
Glibenclamide, by Merck Group (1 mentions)
Dexamethasone (dex), by Sangon (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
C57bl 6 wild, by Jackson ImmunoResearch (1 mentions)
Caspase 1, by Jackson ImmunoResearch (1 mentions)
Human m csf, by R&D Systems (1 mentions)
Mcc950, by Selleck Chemicals (1 mentions)
Bay11 7082, by Merck Group (1 mentions)
Gp91phox, by Cell Signaling Technology (1 mentions)
Phospho p38 thr180 tyr182, by Cell Signaling Technology (1 mentions)
Phospho jak2 tyr1007 1008, by Cell Signaling Technology (1 mentions)
Na k atpase α1, by Cell Signaling Technology (1 mentions)
Ruxolitinib, by Selleck Chemicals (1 mentions)
Cm h2dcfda, by Thermo Fisher Scientific (1 mentions)
Phospho jnk thr183 tyr185, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
13 protocols using apocynin
1
Murine Macrophage Differentiation and Activation
2
Myricitrin Modulates Inflammatory Signaling
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Myricitrin was obtained from Aladdin Industrial Corporation (Shanghai, China). Monoclonal and polyclonal antibodies against iNOS, COX-2, JNK, phospho-JNK (Thr183/Tyr185), p38, phospho-p38 (Thr180/Tyr182), ERK1/2, phospho-ERK1/2 (Thr202/Tyr204), JAK1, phospho-JAK1 (Tyr1022/1023), JAK2, phospho-JAK2 (Tyr1007/1008), STAT1, phospho-STAT1 (Tyr701), STAT3, phospho-STAT3 (Tyr705), phospho-STAT3 (Ser727), TBP, gp91phox, Na/K ATPase-α1, and GAPDH were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibody to p47phox was obtained from Santa Cruz Biotechnology (CA, USA). All secondary antibodies used for western blotting were purchased from LI-COR Biosciences (Lincoln, NE, USA). LPS (from Escherichia coli 0111:B4), NAC, and DAPI were obtained from Sigma-Aldrich (St. Louis, MO, USA). CCK-8 was purchased from KeyGen Biotech (Nanjing, JS, China). CM-H2DCFDA was obtained from Invitrogen (Carlsbad, CA, USA). Ruxolitinib and apocynin were purchased from Selleck Chemicals (Houston, TX, USA). All ELISA kits were purchased from R&D Systems China Co. Ltd. (Shanghai, China).
3
Comprehensive Antibody Panel for Autophagy
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Anti-S. pneumoniae (SSI), anti-GFP (Cell Signaling), anti-Myc (9B11, Cell Signaling), anti-Flag (Wako), anti-Galectin-3 (SINO BIOLOGICAL), anti-Calcoco2 (Proteintech for WB), anti-NDP52 (Gene Tex (GTX115378) for IF), anti-K63 linked Ub (clone Apu3, EMD Millipore), anti-LC3, p62, RFP, Atg16L1, ubiquitin (MBL), and anti-actin (Santa Cruz Biotechnology, Inc.) were used as primary antibodies. An HRP-conjugated goat anti-rabbit or anti-mouse antibodies (Jackson Laboratories) were used as secondary antibodies for immunoblotting. FITC- or TRITC-conjugated goat anti-rabbit or anti-mouse IgG antibodies (Sigma-Aldrich) were used as secondary antibodies for immunostaining. DAPI (4′,6-diamidino- 2-phenylindole, Sigma-Aldrich) was used for DNA staining. LysoTracker DND-99 was purchased from molecular probes. 10 µM rapamycin and 40 µM chloroquine (Selleck chemical), and 30 µM PYR-41 (UBPBio), and 10 mM 3-methyladenine (3-MA, Wako) were used as autophagy inducer or inhibitor. 300 µM Apocynin (Selleck), 10 mM GSH (Cayman Chemical), 2.5 mM NAC (Sigma-Aldrich) were used as antioxidative reagents. All other reagents were purchased from Sigma-Aldrich. All antibodies were used at 1:100 for immunofluorescence staining and 1:1000 for western blotting.
4
Investigating Leptin's Cardioprotective Mechanisms
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The H9C2 rat cardiomyoblasts were purchased from the Cell Resource Center of Peking Union Medical College. The cells were cultured in a modified culture medium containing 10% fetal bovine serum and 1% penicillin–streptomycin at 37 °C with 5% CO2 and 95% air. The cell growth state was monitored daily, and the medium was changed every 2 days. Cells reaching 80% to 90% confluence were subcultured at a 1:3 dilution for further proliferation. Cells attaining 50% to 60% confluence were cultured in serum‐free medium for 12 hours before intervention. The H9C2 rat cardiomyoblasts were pretreated for 1 hour with the leptin antagonist leptin tA (PROSPEC), the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor apocynin (Selleck Chemicals LLC), the protein kinase C (PKC) inhibitor GF109203X (Selleck Chemicals LLC), the reactive oxygen species (ROS) scavenger N‐acetyl‐L‐cysteine (NAC; Sigma Chemical), the activator protein 1 (AP‐1) inhibitor SR11302 (Selleck Chemicals LLC), the mitochondrial‐targeted antioxidant MitoQ (Cayman Chemical), or the mitochondrial permeability transition pore (mPTP) blocker cyclosporine A (Selleck Chemicals LLC), before the treatment with leptin (PeproTech), control rat EAT‐CM, or MetS rat EAT‐CM for 48 hours.
5
Isorhamnetin Regulates Mitochondrial Dynamics
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Isorhamnetin was purchased from Must Biotechnology (Chengdu, China); chloroquine from Sigma-Aldrich; Mn-TBAP from Focus Biomolecules; apocynin was purchased from Selleck Chemicals (Shanghai, CA). The antibodies against cleaved-caspase 3 (9661), pro-caspase 3 (9668S), p62 (5114S), phospho-CamkII (T286, 12,716), phospho-Drp1 (S616, 3455), phospho-Drp1 (S637, 4876), and Drp1 (8570) were purchased from Cell Signaling Technology; PARP (1078–1) was purchased from Epitomics; β-actin (A1978) and LC3 (L754S) from Sigma-Aldrich; Bax (510804), Cleaved-PARP (380374), Bak (380976), NOX2 (381293), NOX4 (380874) and COX IV (200147) from Zen-Bio. Cytochrome. C (13156) and CamkII (5306) were purchased Santa Cruz Biotechnology.
6
Cochlear Explant Culture Protocol
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Mice to be used for cochlear explants were collected between postnatal day 0 (P0) and P1. They were rapidly decapitated and their organs of Corti (OC) were microdissected in ice cold 10% Hank’s Basic Salt solution (HBSS, Gibco) with .05% HEPES (Hyclone) in ultrapure water (Invitrogen) set to a pH of 7.2. Culture plates (50 mm glass bottom dishes, MatTek) were warmed in the incubator at 37° C for 1 hour with a 100 μl mixture of Matrigel (Corning) and DMEM (Gibco) at a ratio of 6:100 by volume coating the center of the dish. After dissection, the tissue was transferred via micro dissecting curette into the Matrigel/DMEM mixture. Excess Matrigel/DMEM mixture was then removed without exposing the tissue to the air, and tissue was left to adhere to the dish for 10 minutes before culture media was added. Culture media was DMEM supplemented with 4% FBS (Hyclone), 1% N2 supplements (R&D Systems), 1 nM apocynin (Selleckchem) and 10 mg/ml ampicillin sulfate (Fisher). Cultures were placed in a 37°C incubator (5% CO2) for 24 hours before receiving EERI, Tyrphostin 23, or fresh media.
7
Chondrocyte Oxidative Stress Response
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Isolated rat chondrocytes were cultured in Hyclone Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum (16000-044, Gibco, Waltham, MA, United States) and 100 U/mL penicillin (Solarbio, Beijing, China). Cells were seeded in a 96-plated well (4 × 103 cells/well in 100 µL of cultured medium) and cultured at 37 °C under a humidified atmosphere of 5% CO2 and 95% air for 12 h before the beginning of the study. Rat chondrocytes were treated with different concentrations of H2O2 (0, 50, 100, and 200 μM). In addition, rat chondrocytes were treated with 100 μM H2O2 and transduced with USP7-silencing vector or USP7 inhibitor 5 μM P22077 (Selleck, Houston, TX, United States) treatment. Moreover, rat chondrocytes were transduced with USP7 expression vector with ROS inhibitor 50 μM apocynin (Selleck) or NOX4 inhibitor 10 μM GLX351322 (Selleck) treatment.
8
ROS Detection in Cell Cultures
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ROS level was detected through either chemiluminescence or flow cytometry. For chemiluminescence assay, cells following treatment (static culture, OSS 30 min, LPS, and TAK-242+OSS 30 min) were gently washed twice with ice-cold PBS, incubated with ROS Fluorescent Probe-DHE (5 μmol/L, Beyotime) at 37°C for 20 min under light-protected conditions and stained with DAPI. Images were obtained with a confocal microscope (ZEISS, German). For flow cytometry analysis, cells that are treated with apocynin, allopurinol, tempol, and rotenone were applied with OSS; after that, cells were trypsinized, resuspended, and incubated with ROS Fluorescent Probe-DHE (5 μmol/L, Beyotime) for analysis. At least 10,000 events were analyzed, and the intensity of fluorescence was determined using the PE-A channel. Data were analyzed using FlowJo V10.3.0 software. LPS was purchased from Sigma-Aldrich (St. Louis, USA). TAK-242 was from MedChemExpress (Shanghai, China). apocynin, allopurinol, tempol, and rotenone were purchased from Selleck (Houston, USA).
9
Investigating Endothelial Cell Signaling
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Dulbecco's Modified Eagle medium (DMEM) and penicillin and streptomycin were purchased from HyClone (Logan, UT, USA). 10% fetal bovine serum (FBS) was obtained from Sijiqing Biological Engineering Materials Co. (Hangzhou, China). Uric acid, telmisartan, diphenylene iodonium (DPI), and ET(B) receptor antagonist BQ788 were purchased from Sigma (St. Louis, MO, USA). Apocynin, PD98059, UO126, and ET(A) receptor antagonist BQ123 were purchased from Selleck Chemicals (Houston, TX, USA).
10
Colon Cancer Cell Cultivation and Compound Treatments
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HCT116 human colon carcinoma cells were obtained from the American Type Culture Collection (Rockville, MD, USA). The cells were cultured at 37 °C in a 5% CO2 atmosphere in McCoy’s 5 A medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. LCA was obtained from Sigma Chemical Co. (St. Louis, MO, USA) and dissolved in dimethyl sulfoxide (DMSO) as 30 mM stock solutions. Metformin was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Bay11-7082 (Bay), diphenyleneiodonium (DPI), PD98059 (PD), JNKi, and SB203580 (SB) from Calbiochem (San Diego, CA, USA) and apocynin from Selleckchem (Houston, TX, USA) were dissolved in DMSO and stored at −80 °C. N-acetyl-L-cysteine (NAC) was purchased from Sigma and was freshly prepared by dissolving in water immediately before use.
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