CYP1A2, CYP2B6 and CYP3A4 activity was measured by using P450-Glo™ CYP1A2 (#V8421), CYP2B6 (#V8321) and CYP3A4 (#V9001) Assay systems (Promega) according to manufacturer’s protocols. Results were calculated against the number of viable cells in the culture which was determined by using CellTiter-Glo 3D Cell Viability Assay (Promega) according to manufacturer’s instructions.
Cyp3a4
Manufactured by Promega
Sourced in United States
CYP3A4 is a recombinant human cytochrome P450 enzyme. It is a key enzyme involved in the metabolism of many drugs and other xenobiotics.
Automatically generated - may contain errors
Lab products found in correlation
Cyp2d6, by Promega (2 mentions)
Cyp2c9 p450 glo, by Promega (2 mentions)
P450 glo cyp1a2, by Promega (1 mentions)
Cyp2b6, by Promega (1 mentions)
Celltiter glo 3d cell viability assay, by Promega (1 mentions)
Quinidine, by Merck Group (1 mentions)
Ketoconazole, by Merck Group (1 mentions)
Gentest pre coated pampa plate system, by Corning (1 mentions)
Acetaminophen, by Merck Group (1 mentions)
Nicotine, by Merck Group (1 mentions)
Artemisinin, by Cayman Chemical (1 mentions)
Deoxyartemisinin, by LGC (1 mentions)
P450 glo cyp2b6, by Promega (1 mentions)
5 protocols using cyp3a4
1
Measuring CYP450 Enzyme Activities
2
Evaluating CYP450 Enzyme Inhibition
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The potential drug–drug interactions were
predicted using CYP3A4, CYP2D6, and CYP2C9 P450-Glo assays purchased
from Promega (Madison, WI, USA) according to a manufacturer’s
protocol. PF9601N, ASS234, contilisant, and selegiline were tested
in triplicate at four concentrations (0.1, 1, 10, and 25 μM).
As the reference inhibitors, ketoconazole (KE), quinidine (QD), and
sulfaphenazole (SE) were used (Sigma-Aldrich, St. Louis, MO, USA)
for CYP3A4, CYP2D6, and CYP2C9, respectively.
predicted using CYP3A4, CYP2D6, and CYP2C9 P450-Glo assays purchased
from Promega (Madison, WI, USA) according to a manufacturer’s
protocol. PF9601N, ASS234, contilisant, and selegiline were tested
in triplicate at four concentrations (0.1, 1, 10, and 25 μM).
As the reference inhibitors, ketoconazole (KE), quinidine (QD), and
sulfaphenazole (SE) were used (Sigma-Aldrich, St. Louis, MO, USA)
for CYP3A4, CYP2D6, and CYP2C9, respectively.
3
ADME-Tox Profiling of Compound AS-1
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The ADME-Tox parameters including permeability, metabolic stability, DDIs, and hepatotoxicity were carried out as described previously [9 (link)–11 (link), 13 (link), 47 (link)]. The ability of AS-1 to passively penetrate through the biological membranes was estimated by Gentest Pre-coated PAMPA Plate System (Corning, Tewksbury, MA) and expressed as the permeability coefficient Pe. The human metabolism of the compound AS-1 was studied using human liver microsomes (HLMs) provided by Sigma-Aldrich. The potential DDIs were predicted by luminescent CYP3A4, CYP2D6, and CYP2C9 P450-Glo assays (Promega, Madison, WI). The respective strong CYP’s inhibitors, ketoconazole (KE, half maximal inhibitory concentration (IC50) = 0.14 μM), quinidine (QD, IC50 = 0.01 μM), and sulfaphenazole (SE, IC50 = 0.08 μM), were used as the references. The hepatic safety of AS-1 was estimated here using hepatoma HepG2 cell growth. The cells were seeded in a 96-well plate and incubated in the presence of AS-1 at the concentration range 0.1–100 μM. One micromolar of cytostatic drug doxorubicin (DX) and 10 μM of mitochondrial toxin carbonyl cyanide 3-chlorophenylhydrazone (CCCP) were used as the references.
4
Liver-on-Chip Cytochrome P450 Induction
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To form the liver-on-a-chip model, the harvested spheroids were mixed with the EC gel precursor and then injected into the microfluidic chip. The medium was collected every other day for the quantification of secreted albumin (Bethyl) and nitrogen fixation (Invitrogen). To induce cytochrome P450, acetaminophen (Sigma) and nicotine (Sigma) were mixed directly into the culture medium on day 6. After a 48 h induction, CYP3A4 (Promega) was quantified following the manufacturer instructions.
5
In Vitro Cytokine and CYP450 Assays
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Chemicals and reagents were from Sigma Aldrich (St. Louis, MO, USA) unless otherwise stated. Artemisinin was from Cayman Chemical (Ann Arbor, MI, USA); deoxyArtemisinin from Toronto Research Chemicals (North York, ON, Canada). Lipopolysaccharide was Escherichia coli serotype O111:B4 and dissolved in sterile water at 1 mg/mL before storage at −20 °C. LEGEND MAXTM Enzyme-linked immunosorbent assay (ELISA) kits for rat TNF-α and IL-6 were from BioLegend (San Diego, CA, USA). ELISA kits for rat IL-10 were from Thermo Fisher Scientific (Waltham, MA, USA). P450-Glo CYP2B6 and CYP3A4 kits were from Promega (Madison, WI, USA). Human liver microsomes (HLMs) from a 200-donor pool of male and female donors were from Sekisui XenoTech (Kansas City, KS, USA).
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