CYP450 enzyme inhibition was tested using P450-gloTM assay kits (Promega Corporation) according to the manufacturer’s instructions. Luminescence was measured using an Infinite M1000 Pro (Tecan) after 20 min of stabilization with a luciferin detection reagent.
P450 glotm assay kit
Manufactured by Promega
Sourced in United States
The P450-GloTM Assay Kit is a luminescent-based assay designed to measure the activity of cytochrome P450 enzymes. The kit provides reagents and protocols for performing P450 activity assays in a convenient and high-throughput format.
Automatically generated - may contain errors
Lab products found in correlation
Infinite m1000 pro, by Tecan (1 mentions)
Varioskan lux multimode microplate reader, by Thermo Fisher Scientific (1 mentions)
Rifampicin, by Merck Group (1 mentions)
Rifampicin solution, by Merck Group (1 mentions)
Omeprazole solution, by Merck Group (1 mentions)
Phenobarbital solution, by Merck Group (1 mentions)
Synergy h1 hybrid reader, by Agilent Technologies (1 mentions)
5 protocols using p450 glotm assay kit
1
Cytochrome P450 Enzyme Inhibition Assay
2
CYP1A2 and CYP3A4 Enzyme Assays in HepaRG Cells
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CYP1A2 and CYP3A4 enzyme activities in differentiated HepaRG monolayers were determined using luminescence-based P450-GloTM assay kits supplied by Promega Corporation (Madison, WI, USA) were used following the 48 h exposures. Reagents were prepared according to the manufacturer's instructions. The protocol for the lytic P450-Glo™ assays using cultured cells in monolayers was followed. Cell monolayers were washed twice with phosphate-buffered saline (PBS), then 50 µL of medium containing 3 µM luciferin-IPA (CYP3A4) or PBS (supplemented with 3 mM salicylamide) containing 6 µM luciferin-1A2 was added to all wells and incubated 37 °C, 5% CO2 for 60 min. Next, an equal volume of luciferin detection reagent was added to the wells and briefly mixed on a plate shaker to form a lysate. The plate was allowed to equilibrate to room temperature for 15 min, then the luminescence signal (RLU) was read directly from the plates using a Varioskan Lux multimode microplate reader (Thermo Fisher, Waltham, MA). The net signals were calculated by subtracting background luminescence values from background wells (no-cells) from the treatment and solvent control values. The percent change was determined by dividing net treated values by net untreated values and multiplying by 100.
3
Measuring CYP3A4 Activity in hEHs
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For the determination of CYP3A4 enzyme activity, hEHs were induced with 25 μM rifampicin (Sigma-Aldrich, R3501,Saint Louis, USA) for 48 h. CYP3A4 enzyme activities were measured 3 h after the incubation with subtype-specific substrate using a commercial P450-GloTM Assay Kit (Promega Corporation, Madison, USA). Luciferase activities were then detected by a multi-detection microplate reader, and the results were normalized with cell number.
4
Cytochrome P450 Enzyme Activity Assay
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The Cytochrome P450 enzymes activity was performed using the P450-GloTM Assay Kit (Promega, Madison, WI) according to the manufacturer’s instructions. We tested the activity of different P450 enzymes, in particular the CYP2B6 (P450-Glo CYP2B6 – V8321/2 – Promega, Madison, WI), CYP3A4 (P450-Glo CYP3A4 (Luciferin-IPA) – V9001/2 – Promega, Madison, WI), and the CYP1A2 (P450-Glo CYP1A2 Induction/Inhibition – V8421/2 – Promega, Madison, WI) by incubating them with different inducers. For the CYP2B6 activity assay, undifferentiated hiPSC, primary hepatocytes and differentiated HLCs were incubated with basal medium containing 1000 μM Phenobarbital solution (Sigma), or DMSO (0.1%) for 48 hours. For the CYP3A4 activity assay, undifferentiated hiPSC, primary hepatocytes and differentiated HLCs were incubated with basal medium containing 20 μM Rifampicin solution (Sigma), or DMSO (0.1%) for 48 hours. For the CYP1A2 activity assay, undifferentiated hiPSC, primary hepatocytes and differentiated HLCs were incubated with basal medium containing 50 μM Omeprazole solution (Sigma), or DMSO (0.1%) for 48 hours. Measurement of the activity of each enzyme was performed by reading the luminescence using a luminometer (Synergy H1 Hybrid Reader - Biotek) according to the manufacturer’s instructions. All the experiments were performed in triplicate.
5
Quantifying CYP Enzyme Activities
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To measure the activity of cytochrome P450 (CYP)-3A4, -2B6, -2C9, and -1A2 in both groups, lytic assays were performed by using a P450-GloTM assay kit (V8802, V8322, and V8792, respectively; Promega) based on the manufacturer’s instructions. The LOs and BLOs derived from hESCs were treated with basal media containing rifampicin (25 μM), valproate (100 μM), phenobarbital (100 μΜ), and omeprazole (100 µM) as CYP-3A4, -2B6, -2C9, and -1A2 inducers, respectively, or DMSO (0.1%) as the inducer solvent. The activity of each enzyme was measured by reading its luminescence using a luminometer (SpectraMax i3x), according to the manufacturer’s protocol.
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