PHH were seeded into 96-well plates and cultured for 1d. P450-Glo CYP3A4 Assay from Promega (Madison, WI, USA) was used for a non-lytic reaction according to the manufacturer’s protocol (n = 3) to assess CYP3A4 activity in PHH. Cells were stimulated with the CYP3A4 inducer dexamethasone [29 (link)] for 6-48h. Stimulation with the solvent ethanol (ETOH, 2% (v/v)) was performed as negative control. After incubation, PHH were exposed to 3μM Luciferin-IPA, here 2% (v/v) Dimethyl sulfoxide (DMSO) was added as an inhibitory control. Luminescence was detected after 1h of incubation by using the FLUOstar Omega from BMG LABTECH (Ortenberg, Germany). Mean relative luminescence signals were determined and net signals were calculated by subtracting the background luminescence signal (no cell control).
P450 glo cyp3a4 assay
Manufactured by Promega
Sourced in United States
The P450-Glo CYP3A4 Assay is a luminescent-based assay that measures the activity of the CYP3A4 enzyme. The assay provides a simple and sensitive method for determining CYP3A4 enzyme activity in various sample types.
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Lab products found in correlation
Fluostar omega, by BMG Labtech (1 mentions)
Fluorescein diacetate, by Merck Group (1 mentions)
A83 01, by Merck Group (1 mentions)
Collagenase type 4, by Worthington (1 mentions)
Transwell insert, by Corning (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Nicotinamide, by Merck Group (1 mentions)
Murine egf, by Thermo Fisher Scientific (1 mentions)
Primocin, by InvivoGen (1 mentions)
Cobalt chloride, by Merck Group (1 mentions)
N acetyl cysteine (nac), by MP Biomedicals (1 mentions)
Alp assay kit, by Abcam (1 mentions)
Potassium phosphate buffer, by Merck Group (1 mentions)
Zafirlukast, by Merck Group (1 mentions)
Rat tail collagen type 1, by Corning (1 mentions)
Bicinchoninic acid bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
12 well polystyrene tissue culture dishes, by Corning (1 mentions)
N n 3 5 difluorophenacetyl l alanyl s phenylglycine t butyl ester dapt, by Apexbio (1 mentions)
Human collagen type 1, by Advanced BioMatrix (1 mentions)
Gastrin, by AnaSpec (1 mentions)
13 protocols using p450 glo cyp3a4 assay
1
Assessing CYP3A4 Activity in PHH
2
Hepatocyte Culture Optimization Protocol
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Rat tail type I collagen, Transwell inserts (0.9 cm2 cell culture area, 1.6 × 106 pores/cm2), 12-well polystyrene tissue culture dishes, 12-well polycarbonate plates and Transwell inserts (1.12 cm2 cell culture area, 1.6 × 108 pores/cm2) were purchased from Corning (Corning, NY). SB202190 was from Selleckchem (Houston, TX). Collagenase (type IV) was purchased from Worthington Biochemicals (Lakewood, NJ). Y-27632 and N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) were purchased from ApexBio Technology (Houston, TX). Human collagen (type I) was obtained from Advanced Biomatrix (Carlsbad, CA). The ALP assay kit was from AbCam (Cambridge, MA). Gastrin was from Anaspec (Freemont, CA). UGT-Glo™ Assay and P450-Glo™ CYP3A4 Assay were obtained from Promega (Madison, WI). N-acetyl cysteine was purchased from MP Biomedicals (Santa Ana, CA). Murine EGF was procured from Peprotech (Rock Hill, NJ). Primocin was purchased from InvivoGen (San Diego, CA). Nicotinamide, L-alanine p-nitroanilide, fluorescein diacetate, loperamide, zafirlukast, cobalt chloride, potassium phosphate buffer, bovine serum albumin, and A83–01 were acquired from Sigma-Aldrich (St. Louis, MO). All other reagents and Bicinchoninic Acid (BCA) Protein Assay Kit were purchased from Thermo Fisher Scientific (Waltham, MA).
3
Cryopreserved 3D Spheroid CYP Assay
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Three freeze/thaw spheroids, frozen
in cryovials as a suspension with CPA solution containing MEM base
media supplemented with 10% (v/v) FBS, 10% (v/v) DMSO, and with or
without 20 mg mL–1 of the polyampholyte, were transferred
into a white opaque 96-well plate. Spheroids were washed either 3-,
5- or 7-days post-thaw with 1× phosphate-buffered saline (PBS;
Merck, Gillingham, UK). The PBS was replaced with a culture medium
containing a luminogenic CYP substrate, CYP3A4/Luciferin-IPA (3 μM,
50 μL, 1 h) provided by a Promega P450-Glo CYP3A4 assay. The
CYP substrate was also added to empty wells as a background measurement.
The culture medium containing the CYP substrate (25 μL) was
transferred to an opaque white 96-well plate and a luciferin detection
reagent (25 μL) was added for 20 min at RT. Luminescence was
measured on a Tecan Spark plate reader (Tecan, Switzerland). The CYP
activity of non-frozen spheroids was also measured for comparison.
in cryovials as a suspension with CPA solution containing MEM base
media supplemented with 10% (v/v) FBS, 10% (v/v) DMSO, and with or
without 20 mg mL–1 of the polyampholyte, were transferred
into a white opaque 96-well plate. Spheroids were washed either 3-,
5- or 7-days post-thaw with 1× phosphate-buffered saline (PBS;
Merck, Gillingham, UK). The PBS was replaced with a culture medium
containing a luminogenic CYP substrate, CYP3A4/Luciferin-IPA (3 μM,
50 μL, 1 h) provided by a Promega P450-Glo CYP3A4 assay. The
CYP substrate was also added to empty wells as a background measurement.
The culture medium containing the CYP substrate (25 μL) was
transferred to an opaque white 96-well plate and a luciferin detection
reagent (25 μL) was added for 20 min at RT. Luminescence was
measured on a Tecan Spark plate reader (Tecan, Switzerland). The CYP
activity of non-frozen spheroids was also measured for comparison.
4
Cytochrome P450 3A4 Activity Assay
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Cytochrome P450 3A4 (CYP3A4) activity was analyzed using P450-Glo™ CYP3A4 Assay (Promega). Briefly, a pro-luciferin substrate was added to the cell culture medium and incubated for 1 h at 37 °C. Aliquots of supernatants were collected and incubated with the luminogenic detection reagent before luminescence reading using TECAN® plate reader. All samples were run in triplicate.
5
Evaluating miRNA Effects on CYP3A4
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Cryopreserved human hepatocytes were thawed and plated onto collagen-coated 96 wells at a cell density of 0.7 × 105 cells/ml in 0.1 mL/well. After 12 hours, the culture medium was changed with serum-free medium and transfection of miRNA mimics and inhibitors was conducted. After the 48 h treatment period, CYP3A4 enzyme activity in cryopreserved human hepatocyteswas determined using the P450-Glo™ CYP3A4 assay (Promega, USA), which contained the substrate luciferin isopropyl acetal (luciferin-IPA) and luciferin detection reagent, following the manufacturer’s protocol.
6
Quantification of Secreted Albumin and CYP3A4 Activity
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Secreted albumin was quantified in the culture supernatant with the Human Albumin Enzyme-Linked Immunosorbent Assay (ELISA) Kit (Bethyl laboratories, Montgomery, TX, USA), according to the manufacturer’s instructions, at an absorbance of A450 nm (TriStar Multimode Reader LB942) with a reference of A620 nm.
Cytochrome P450 oxidase 3A4 (CYP3A4) activity was determined with the P450-Glo CYP3A4 Assay (Promega, Mannheim, Germany), according to the manufacturer’s instructions. Briefly, CYP3A4 substrate Luciferin-PFBE (50 µM) was added on cell-laden 3D constructs and incubated at 37 °C, 5% CO2. After 4 h, CYP3A4-mediated conversion of Luciferin-PFBE substrate to luciferin, which is secreted from the printed HepaRG cells, was determined. The supernatant was incubated with Luciferin detection reagent and the luminescence was measured (TriStar Multimode Reader LB942). Luminescence values were normalized to lysis controls. For cell lysis, cell-laden 3D constructs were incubated in culture medium supplemented with 10% Triton-X-100.
Cytochrome P450 oxidase 3A4 (CYP3A4) activity was determined with the P450-Glo CYP3A4 Assay (Promega, Mannheim, Germany), according to the manufacturer’s instructions. Briefly, CYP3A4 substrate Luciferin-PFBE (50 µM) was added on cell-laden 3D constructs and incubated at 37 °C, 5% CO2. After 4 h, CYP3A4-mediated conversion of Luciferin-PFBE substrate to luciferin, which is secreted from the printed HepaRG cells, was determined. The supernatant was incubated with Luciferin detection reagent and the luminescence was measured (TriStar Multimode Reader LB942). Luminescence values were normalized to lysis controls. For cell lysis, cell-laden 3D constructs were incubated in culture medium supplemented with 10% Triton-X-100.
7
Hepatocyte CYP gene expression and activity
8
Quantifying CYP3A4 Activity in FL-MSCs
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We used the P450-Glo CYP3A4 assay where the measurement of luciferase reports the P450 cytochrome activity (Promega). Briefly, FL-MSCs were incubated with IMDM containing 50 μmol luciferin-IPA, at 37°C. Three hours later, 50 μL of medium was transferred in a 96-well opaque white plate (Costar), mixed with 50 μL of luciferin detection reagent and incubated for 20 min at room temperature. Luminescence was measured using a Victor3 luminometer.
9
CYP3A4 Activity Quantification Protocol
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CYP3A4 activity in tissue was determined with a P450-Glo CYP3A4 Assay (Promega). A standard curve was generated with D-luciferin (Promega). The luminescence was measured with a Fluoroskan Ascent FL (ThermoScientific).
10
Quantifying CYP3A4 Inhibition by Pantoprazole
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Inhibition of CYP3A4 by pantoprazole was quantified with the P450-Glo™ CYP3A4 Assay (Promega Corporation, Madison, WI, USA) using a membrane preparation containing recombinant human CYP3A4 according to the manufacturer’s instructions. This assay is based on the conversion of the luminogenic CYP3A4 substrate luciferin-IPA to luciferin by CYP3A4 and was conducted as described previously [20 (link)]. Eight concentrations of pantoprazole (0.05–100 µM) were investigated in triplicate, and the experiment was conducted in quadruplicate. Data were analysed and half maximal inhibitory concentrations (IC50) were calculated with GraphPad Prism Version 10.0 (GraphPad Software, San Diego, CA, USA) using the four-parameter fit (sigmoidal concentration–response curves with variable slope).
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