Human cancer cell lines MCF7, MDA-MB-231, HCCLM3 and murine cancer cell lines 67NR, 4T1, CMT93 were obtained from ATCC. All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) with 10% fetal bovine serum Gibco), 1% penicillin and streptomycin, and they were maintained at 37 °C and 5% CO2. MCherry and luciferase-expressing 4T1 cells were established by transfection with lentivirus expressing mCherry and luciferase. The sequences of sgRNA and shRNA referred above were listed in Supplementary Tables 1 –3 . The potassium oxalate (20 μM, Aladdin), DNase I (100 U/ml, Aladdin), CCPST (200 μM, Key organics), PMA (10 nM, MedChemExpress), apocynin (10 μM, MedChemExpress), SB 203580 (500 nM, MedChemExpress) and CU CPT 4a (27 μM, Tocris) were used for cell treatment.
Apocynin
Manufactured by MedChemExpress
Sourced in United States
Apocynin is a natural compound that acts as an inhibitor of the NADPH oxidase enzyme complex. It is a research tool used in scientific studies to investigate the role of NADPH oxidase-derived reactive oxygen species in various biological and pathological processes.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Phorbol myristate acetate (pma), by MedChemExpress (2 mentions)
Cu cpt 4a, by Bio-Techne (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Sb203580, by MedChemExpress (2 mentions)
Mcf 7, by American Type Culture Collection (1 mentions)
Vigabatrin, by MedChemExpress (1 mentions)
Cgp35348, by Merck Group (1 mentions)
Yohimbine, by MedChemExpress (1 mentions)
Db camp, by MedChemExpress (1 mentions)
Sq22536, by MedChemExpress (1 mentions)
Tempol, by MedChemExpress (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Huvecs, by Merck Group (1 mentions)
Ha vsmc, by American Type Culture Collection (1 mentions)
Aicar, by Merck Group (1 mentions)
Huvecs, by American Type Culture Collection (1 mentions)
5 protocols using apocynin
1
Establish Human and Murine Cancer Cell Lines
2
Neutrophil Activation Assay
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For each well in 48 well plates, 5 × 106 Neutrophils were seeded in 200 μl DMEM containing high glucose (5.5, 15, 25, or 35 mM) and PMA (100 nM), and incubated at 37°C with 5% CO2 for 120 min. In some experiments, neutrophils were pretreated with diphenyleneiodonium (DPI, 5 μM. MedChemExpress, USA) and Apocynin (10 μM. MedChemExpress, USA), the most widely used NADPH oxidase inhibitors, blocking for 30 min before stimulation.
3
Characterizing Neuroinflammatory Modulators
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Tempol, apocynin, db-cAMP, SQ22536, vigabatrin, gabazine, yohimbine and clonidine were obtained from MedChemExpress (Monmouth Junction, NJ, USA). LPS, CGP35348, DEX and DETC were purchased from Sigma (St Louis, MO, USA). Rp-cAMP was purchased from Beyotime (Shanghai, China). apocynin and CGP35348 were dissolved in PBS containing 1% DMSO and the vehicle was used as a control. Other chemicals were dissolved in PBS. PBS was used as a control.
4
HUVEC-Mediated Vascular Smooth Muscle Regulation
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Human umbilical vein endothelial cells (HUVECs, ATCC) were cultured according to manufacturer’s instructions. Cells of passage 6–8 were used for experiments. Before treatment, HUVECs were serum deprived in 0.5% FBS medium for 6 h. When subjected to TSH (Sigma), CsA (Solarbio), AICAR(Sigma) or Apocynin (MedChem Express), cells were cultured in 2.5% FBS medium.
HUVECs were treated with TSH (2 μmol/L) or vehicle for 24 h. After the incubation period, the medium was collected as conditioned medium (CM). Human aortic smooth muscle cells (HA-VSMCs, ATCC) of passage 5–7 were allowed to adhere for 24 h. After reaching 70–80% confluence, the cells were synchronized in serum free medium for 6 h and then cultured in 2.5% FBS medium, 2.5% FBS medium containing 2 μmol/L TSH or CM for 24 h.
HUVECs were treated with TSH (2 μmol/L) or vehicle for 24 h. After the incubation period, the medium was collected as conditioned medium (CM). Human aortic smooth muscle cells (HA-VSMCs, ATCC) of passage 5–7 were allowed to adhere for 24 h. After reaching 70–80% confluence, the cells were synchronized in serum free medium for 6 h and then cultured in 2.5% FBS medium, 2.5% FBS medium containing 2 μmol/L TSH or CM for 24 h.
5
Culturing and Transducing Cancer Cell Lines
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