Apocynin

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Apocynin is a naturally occurring organic compound that is commonly used as a laboratory reagent. It is a potent inhibitor of the NADPH oxidase enzyme complex, which plays a role in the production of reactive oxygen species. Apocynin is often utilized in research applications where the modulation of oxidative stress and inflammation is of interest.

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Lab products found in correlation

Mito tempo, by Santa Cruz Biotechnology (2 mentions) Sb203580, by Santa Cruz Biotechnology (2 mentions) Sp600125, by Fujifilm (2 mentions) Catalase, by Merck Group (2 mentions) Celllabs quanta flow cytometer, by Beckman Coulter (2 mentions) Accutase, by GE Healthcare (2 mentions) β actin, by Santa Cruz Biotechnology (2 mentions) Nsc87877, by Cayman Chemical (1 mentions) U0126, by Fujifilm (1 mentions) Doxorubicin dox, by Merck Group (1 mentions) Cisplatin, by Fujifilm (1 mentions) Anisomycin, by Fujifilm (1 mentions) Etoposide, by Santa Cruz Biotechnology (1 mentions) Wortmannin, by Cayman Chemical (1 mentions) N acetylcysteine, by Fujifilm (1 mentions) Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions) Streptomycin, by Thermo Fisher Scientific (1 mentions) Newborn calf serum, by Thermo Fisher Scientific (1 mentions) Penicillin g, by Thermo Fisher Scientific (1 mentions) Thapsigargin, by Merck Group (1 mentions)

16 protocols using apocynin

Assessing Macrophage NADPH Oxidase Role in Tumor Microenvironment

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To assess indirectly the role of macrophage NADPH oxidase in the generation of physiological levels of ROS in co-culture microenvironment, cells were treated with 300 µM of apocynin, a NADPH oxidase inhibitor that acts by blocking the assembly of NADPH oxidase subunits (Santa Cruz Biotechnology) for 48 h. Moreover, to determine whether NADPH oxidase-generated oxidative stress can modulate C26 cell proliferation, tumor angiogenesis and inflammation, all assays described above were performed after co-culture incubation with apocynin.
Statistical analysis. Data from different experiments are expressed as mean ± standard deviation (SD). The differences between different protumor processes under standard and co-culture conditions were evaluated using unpaired t-test.
The differences between the production of angiogenic proteins in cells from standard culture and co-culture were analyzed by two-way ANOVA with Bonferroni correction for multiple comparisons. Correlations between different parameters were evaluated using Pearson correlation coefficient, r. All statistical analyses were performed using GraphPad Prism version 6 for Windows, GraphPad Software (San Diego, CA, USA). A P-value <0.05 was considered significant.

Luput L., Licarete E., Sesarman A., Patras L., Alupei M.C, & Banciu M. (2017). Tumor-associated macrophages favor C26 murine colon carcinoma cell proliferation in an oxidative stress-dependent manner. Oncology reports, 37(4).

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Diverse Pharmacological Inhibitors Assay

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Doxorubicin (Dox) was purchased from Sigma. Cisplatin, anisomycin, propyl gallate (PG), SP600125, N-acetylcysteine (NAC) and U0126 were purchased from Wako. SB203580, Etoposide, Apocynin (Apo) and mitoTEMPO (MT) were purchased from Santa Cruz. NSC87877 and wortmannin were purchased from Cayman.

Hirata Y., Inoue A., Suzuki S., Takahashi M., Matsui R., Kono N., Noguchi T, & Matsuzawa A. (2020). trans-Fatty acids facilitate DNA damage-induced apoptosis through the mitochondrial JNK-Sab-ROS positive feedback loop. Scientific Reports, 10, 2743.

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Calcium Signaling and Oxidative Stress

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M-199, 100 IU/mL penicillin G and 100 µg/mL streptomycin were purchased from Gibco. Newborn calf serum was purchased from Life Technology. BK, thapsigargin, VAS2870, and HOE-140 were purchased from Sigma-Aldrich. KN-93, W-7 hydrochloride, Trolox, and Phos-tag were purchased from Wako. Fura-2/AM and BAPTA/AM were purchased from Dojindo. C-DCDHF-DA was purchased from Invitrogen. EzBlock Chemi™ and Ez RIPA Lysis Kit™ were purchased from ATTO. Anti-NOX2, 3, and 5 antibodies were purchased from Proteintech. Apocynin and horseradish peroxidase-conjugated secondary antibody were purchased from Santa Cruz Biotechnology.

Sakurada R., Odagiri K., Hakamata A., Kamiya C., Wei J, & Watanabe H. (2019). Calcium Release from Endoplasmic Reticulum Involves Calmodulin-Mediated NADPH Oxidase-Derived Reactive Oxygen Species Production in Endothelial Cells. International Journal of Molecular Sciences, 20(7), 1644.

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Uric Acid Induced Oxidative Stress Assessment

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Cells were seeded onto 35-mm confocal dishes (with a cover glass) and classified into control, normal uric acid (UA), and high uric acid (HUA) groups (n = 3). Cells in the UA and HUA groups were cultured in medium containing 300 or 600 μM uric acid, respectively, for 24 h followed by incubation with the total oxidative stress indicator chloromethyl derivative dichlorodihydrofluorescein diacetate (CM-H₂DCFDA, Beyotime, Nanjing China, 5 μM) for 30 min in the dark at 37 °C. After three washes in PBS, green fluorescence was visualized using a laser scanning confocal microscope at an excitation wavelength of 488 nm and an emission wavelength of 515 nm.
Before HUA treatment, the HUA group was pretreated with 0.5 mM apocynin (Santa Cruz Biotechnology) or 0.1 μM epalrestat (Santa Cruz Biotechnology). Pyocyanin (100 μM; Sigma) was used as a positive control in the comparisons of ROS production.

Huang Z., Hong Q., Zhang X., Xiao W., Wang L., Cui S., Feng Z., Lv Y., Cai G., Chen X, & Wu D. (2017). Aldose reductase mediates endothelial cell dysfunction induced by high uric acid concentrations. Cell Communication and Signaling : CCS, 15, 3.

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Quantifying Frameshift Mutation Rates

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5 × 10³–1 × 10⁴ EGFP-negative HCT116 + chr3 or HCEC-1CT frameshift-reporter cells were sorted into 24-well plates on a FACSAria cell sorter using CloneCyt Plus software (BD Biocsiences, San Jose, CA). Twenty-four hours later, freshly isolated PMNs were added together with 0.5 nM PMA at effector:target ratios ranging from 0:1 (control) to 75:1. After 24 h, PMN debris and medium were removed by washing with PBS. Fresh culture medium was added, and target cells were grown for another 6 days. For some experiments PMNs were treated with superoxide dismutase (SOD; Sigma, S7571), apocynin (Santa Cruz, sc-203321) or catalase (Sigma, C3155) at indicated concentrations. After 24 h, medium was renewed.
Target cells were detached using 160 μl accutase (PAA Laboratories GmbH, Linz, Austria) and analyzed on a CellLabs Quanta flow cytometer (Beckman Coulter, Brea, CA). Flow cytometric analysis and calculation of mutation rates (MRs) were performed as described previously (18 (link),20 (link)). Data are presented as fold changes to control cells (±95% confidence interval). Also for HCEC-1CT, although mixed clones, fold changes for MRs were calculated, assuming one plasmid insertion in each clone (18 (link)).

Granofszky N., Lang M., Khare V., Schmid G., Scharl T., Ferk F., Jimenez K., Knasmüller S., Campregher C, & Gasche C. (2018). Identification of PMN-released mutagenic factors in a co-culture model for colitis-associated cancer. Carcinogenesis, 39(2), 146-157.

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Quantifying Frameshift Mutation Rates

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Mitochondrial Dysfunction and Oxidative Stress

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Cell culture medium (DMEM), fetal bovine serum (FBS) and antibiotics were procured from Cellgro® (Manassas, VA). MitoSOX™, Mito Tracker® Green, CM-H2DCFDA and Hoechst 33342 were purchased from Life Technologies (Grand Island, NY). 1,2-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid tetrakis (acetoxymethyl) ester (BAPTA-AM) (Sigma Aldrich, St. Louis, MO). N-acetyl L-cysteine (NAC) (Sigma Aldrich, St. Louis, MO). L- N^G nitroarginine methyl ester (L-NAME, Sigma Aldrich, St. Louis, MO). N-(3-(Aminomethyl)benzyl)acetamidine (1400W) (Sigma Aldrich, St. Louis, MO). Mito-Vitamin E and Mito-CoQ were synthesized in the laboratory of Dr. Kalyanaraman. Mito-TEMPO (Cayman Chemical company, Ann Arbor, MI). Apocynin (Santa Cruz Biotechnology, Santa Cruz, CA). All other chemicals were purchased from either Sigma Aldrich or Thermo Fisher Scientific (Tustin, CA).

Gangwar R., Meena A.S., Shukla P.K., Nagaraja A.S., Dorniak P.L., Pallikuth S., Waters C.M., Sood A, & Rao R. (2017). Calcium-Mediated Oxidative Stress: a Common Mechanism in Tight Junction Disruption by Different Types of Cellular Stress. The Biochemical journal, 474(5), 731-749.

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High-Throughput Screening of Small Molecule Regulators

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The first screening was conducted using a chemical library (Stem Select^TM Small Molecule Regulators 384-Well Library I, Merck). Each compound was diluted to 10 mmol·L⁻¹ so that the final concentration in the cell culture plate would become 10 μmol·L⁻¹. The compounds used in the experiments other than primary screening were purchased individually: AICAR (Calbiochem), monastrol (Santa Cruz Biotechnology), PARP inhibitor XII (Calbiochem), AMI-5 (Calbiochem), 17b (Calbiochem), IWR-1-endo (Calbiochem), apocynin (Santa Cruz Biotechnology), L-165, 041 (Abcam Limited), linifanib (Cayman Chemical Company), SR18292 (Cayman Chemical Company), FK506 (Cayman Chemical Company) and KN93 (Cayman Chemical Company). These compounds were dissolved in DMSO (Sigma) so that their concentrations were 1 000 times greater than the final concentration.

Ono T., Denda R., Tsukahara Y., Nakamura T., Okamoto K., Takayanagi H, & Nakashima T. (2022). Simultaneous augmentation of muscle and bone by locomomimetism through calcium-PGC-1α signaling. Bone Research, 10, 52.

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Pharmacological Agents for Cell Signaling

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Morphine (M8777-250MG; Sigma-Aldrich), naltrexone (N3136-100MG; Sigma-Aldrich), PDGF-BB (220-BB-050; R&D Systems), PBN (B7263-1G; Sigma-Aldrich), apocynin (sc-203321; Santa Cruz Biotechnology, Inc.), mitoTEMPO (SML0737; Sigma-Aldrich), 4-PBA (50–230-7444; Thermo Fisher Scientific), salubrinal (SML0951; Sigma-Aldrich), wortmannin (W3144; Sigma-Aldrich), 3-MA (M9281; Sigma-Aldrich), and bafilomycin (B1793; Sigma-Aldrich) were obtained from commercial vendors as described.

Cai Y., Yang L., Hu G., Chen X., Niu F., Yuan L., Liu H., Xiong H., Arikkath J, & Buch S. (2016). Regulation of morphine-induced synaptic alterations: Role of oxidative stress, ER stress, and autophagy. The Journal of Cell Biology, 215(2), 245-258.

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Autophagy Regulation in Induced Pluripotent Stem Cells

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iPSCs were always cultured on Matrigel (Corning, 356237) in E8 flex supplemented with primocin (0.1 µg/ml; Invivogen, ANT-PM-05) and low puromycin and G418 concentrations (0.5 µg/ml) at 37°C, 5% CO₂. Medium was refreshed every 2–3 days and cells were passaged 1–2 times per week using an enzyme-free reagent (ReLeSR; Stem Cell Technologies, 05872). For autophagy induction cells were treated with 200 nM rapamycin (Santa Cruz Biotechnology, sc-3504) for 10 min, before medium was refreshed. To block autophagosome formation or lysosome fusion cells were treated with 200 nM wortmannin (Invivogen, tlrl-wtm) or bafilomycin A₁ (Millipore, 19–148), respectively prior to rapamycin treatment. If not mentioned differently, cells were lysed/fixated after 2 h of incubation. For apocynin (Santa Cruz Biotechnology, sc-20332) rescue experiments iPSCs were plated as single cells. The day after plating the cells were treated for 24 h with 200 µM apocynin before cells were fixed. To stimulate ROS production cells were treated for 10 min with 100 µM BSO (Sigma-Aldrich, B2515). After medium was refreshed, cells were incubated for 2 h before lysate preparation or fixation.

Linda K., Lewerissa E.I., Verboven A.H., Gabriele M., Frega M., Klein Gunnewiek T.M., Devilee L., Ulferts E., Hommersom M., Oudakker A., Schoenmaker C., van Bokhoven H., Schubert D., Testa G., Koolen D.A., de Vries B.B, & Nadif Kasri N. (2021). Imbalanced autophagy causes synaptic deficits in a human model for neurodevelopmental disorders. Autophagy, 18(2), 423-442.

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