For CYP activity measurement, “P450-Glo™ Assays” (Promega, Madison, Wisconsin, USA) were used. CYP1A2 activity was detected by Methoxy-Luciferin (Luciferin-ME) turnover (V8772), CYP2B6 was detected by Dimethoxybutyl-Luciferin (Luciferin-2B6) turnover (V8321) and CYP3A4 was detected by Luciferin isopropyl acetal (Luciferin-IPA) turnover (V9001). The reaction was performed according to the Promega “P450-Glo™ Assays” protocol except the CYP reaction time was prolonged to 1 h unless otherwise noted. The reaction temperature was set at 37 °C. The NADPH Regeneration System (V9510) was used for the supply of NADPH during the assay. Three control approaches were performed, one with buffer control, one designed as no template control and one control, using human liver cell microsomes (Gibco™ Human Microsomes, 50 Donors) (Thermo Fisher Scientific, Waltham, Massachusetts, USA) as positive control. Human microsomes were tested at a final concentration of 0.4 mg/mL. If not otherwise noted 5 µL of the microsomal fraction of the cell-free reaction were applied as samples to the activity assay. For response condition adjustments, CYP activities were usually expressed as percentage of the highest CYP activity during the assay. For CYP activity quantification, a standard curve was prepared using beetle luciferin (Promega, Madison, Wisconsin, USA) according to the protocol.
P450 glo assay
Manufactured by Promega
Sourced in United States
The P450-Glo assay is a luminescent-based assay that measures the activity of cytochrome P450 enzymes. It provides a sensitive and quantitative method for determining P450 enzyme activity in vitro.
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Lab products found in correlation
Rifampicin, by Merck Group (4 mentions)
Glutathione s transferase assay kit, by Cayman Chemical (2 mentions)
Endoplasmic reticulum isolation kit, by Merck Group (2 mentions)
Nadph regeneration system, by Promega (2 mentions)
Lumistar optima, by BMG Labtech (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Trypan blue stain, by Thermo Fisher Scientific (2 mentions)
Luciferin pfbe, by Promega (2 mentions)
Luciferin h ege, by Promega (2 mentions)
Hmcpms lot hu8138, by Thermo Fisher Scientific (2 mentions)
Luciferin ipa, by Promega (2 mentions)
Microsome isolation kit, by Abcam (1 mentions)
Glomax explorer, by Promega (1 mentions)
Celltiter glo, by Promega (1 mentions)
Pluronic f 127, by Merck Group (1 mentions)
Lumat lb 9507, by Berthold Technologies (1 mentions)
Infinite f500 plate reader, by Tecan (1 mentions)
Zoledronate, by Merck Group (1 mentions)
Risedronate, by Merck Group (1 mentions)
36 protocols using p450 glo assay
1
CYP Enzyme Activity Measurement
2
Colon and Liver Microsome Assay
3
CYP3A4 Activity Measurement via Luminescence
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CYP3A4 enzymatic activity was measured by the P450-Glo Assay (Promega) luminescent method as manufacturer’s protocol.
4
Measuring CYP2C9 and CYP3A4 Activities
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CYP2C9 and CYP3A4 enzyme activities were assessed by P450‐Glo assay (Promega, Madison, WI, USA), according to the manufacturer's instructions. Differentiated cells were treated with or without 25 μM rifampicin (Sigma‐Aldrich), a PXR agonist, for 72 hrs, and the media were changed on the cells every day. The cells were incubated at 37°C in fresh medium with Luciferin‐H for 4 hrs or Luciferin‐IPA for 60 min. separately. After the incubation, 50 μl of medium was transferred to a 96‐well plate and mixed with 50 μl of luciferin detection reagent to initiate the luminescent reaction. After 60 min. of incubation at 37°C, the luminescence was measured with a luminometer (Envision2104‐0010, Waltham, MA, USA).
5
Isolation and Enzymatic Assays of Colon and Liver Microsomes
6
Assessing CYP2C19 Inhibition Potential
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The inhibition of CYP2C19 activity was determined using the P450-Glo assay (Promega) according to the manufacturer’s instructions. Briefly, purified recombinant human CYP2C19 isozyme was purchased from Promega. The enzyme was incubated with 100 µg/mL of each compound for 30 minutes, followed by the addition of the specific luminescent substrate and the NADPH regenerating system (containing NADP+, glucose-6-phosphate, MgCl2, and glucose-6-phosphate dehydrogenase) provided with the kit. The reaction mixture was incubated at room temperature for 30 minutes followed by the addition of the luciferin detection reagent. The luminescence was measured using a luminometer after incubation with the reagent for 20 minutes.
7
Cytochrome P450 Enzyme Assay
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CYP measurement was performed on day eight following the formation of LBs using the P450-Glo™ assay (Promega), according to the manufacturer’s instructions. Rifampicin with or without ketoconazole or sulfaphenazole was loaded eight hours before the collection of medium for analysis.
8
Hepatic Microsome Isolation and CYP3A4 Assay
9
Quantifying Cytochrome P450 Activities
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CYP 3A and CYP 1A2 activity were measured on HLCs at day 18 by using the P450-Glo™ assay (Promega). CYP 3A (1:20) and CYP 1A2 (1:50) substrates were diluted in hepatocyte maturation medium and added to the HLCs for 5 37 °C in 5% (v/v) CO2, plain medium with substrate was incubated in parallel to subtract background. After incubation, 50 μL of supernatant was collected and mixed with 50 μL of luciferin detection reagent in a white 96-well plate at room temperature in the dark for 20 min. Luminescence was measured using a multiplex plate reader (GloMax Explorer, Promega) and normalized per mg of protein (RLU/ml/mg) quantified by BCA assay.
10
Quantifying CYP activity via P450-Glo assay
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Measurement of the activity of all four CYPs was conducted using the P450-Glo™ assay (Promega, Japan), according to the manufacturer’s instructions. Briefly, cells were seeded in a 96-well plate at a density of 1×104 cells/well. Twenty-four hours later, test compounds and the luminescent substrate included in the kit were added into the culture media. Following 1 hour of incubation, the detection reagent was added, and the luminescence generated by the metabolic substrate within the culture media was measured using an Infinite F500 plate reader (Wako). The inhibitor test conditions utilized in this study are listed in the results.
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