Cell samples were collected in RNAprotect Cell Reagent (Cat. No. 76526, QIAGEN) during the differentiation procedure on days 9, 11, 14, 16, 18, 21, 23, and 25. RNA was extracted from these samples using MagMAX-96 Total RNA Isolation Kit (Cat. No. AM1830, ThermoFisher). cDNA was synthesized by applying High-Capacity cDNA Reverse Transcription Kit (Cat. No. 4368814, ThermoFisher). Real-time quantitative PCR (RT-qPCR) was performed using TaqMan Fast Advanced Master Mix (Cat. No. 4444557, ThermoFisher) for the following markers: KRT19 (Hs00761767_s1, ThermoFisher), CYP2C9 (Hs04260376_m1, ThermoFisher), MIR370 (Hs04231551_s1, ThermoFisher), FLJ22447(Hs01382450_m1, ThermoFisher), and FLJ22763 (Hs01396927_m1). GAPDH (Hs99999905_m1, ThermoFisher) was used as a reference gene, and cDNA synthesized on a cocktail of RNA extracted from different cell types was used as a calibrator.19 (link)
Cyp2c9
Manufactured by Thermo Fisher Scientific
Sourced in United States
CYP2C9 is a recombinant human cytochrome P450 enzyme. It is a key enzyme involved in the metabolism of various drugs and other xenobiotic compounds.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using cyp2c9
1
Temporal Gene Expression Profiling
2
Quantitative RT-PCR Analysis of CAR Targets
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RT-qPCR was used to examine CAR target gene
expression in PHH, HepaRG cells, or in mouse liver samples. Total
RNA, reverse transcription, and qPCR were performed, and mRNA expression
data was analyzed as we have described before.51 (link)All RT-qPCR experiments were performed in triplicate
samples, and data are presented as fold induction to vehicle-treated
cells with the same reagents we have described before.48 (link),52 (link) PCR TaqMan probes for murine genes have been listed in our previous
reports.52 (link) TaqMan probes for CYP3A4, CYP2C9, and CYP2B6 human genes
as well as for reference genes B2M and GADPH genes were obtained from Thermo Fisher: CYP3A4 (Hs00604506_m1), CYP2C9 (Hs02383631_s1), CYP2B6 (Hs04183483_g1), GAPDH (Hs02758991_g1), and B2M (Hs00984230_m1).
expression in PHH, HepaRG cells, or in mouse liver samples. Total
RNA, reverse transcription, and qPCR were performed, and mRNA expression
data was analyzed as we have described before.51 (link)All RT-qPCR experiments were performed in triplicate
samples, and data are presented as fold induction to vehicle-treated
cells with the same reagents we have described before.48 (link),52 (link) PCR TaqMan probes for murine genes have been listed in our previous
reports.52 (link) TaqMan probes for CYP3A4, CYP2C9, and CYP2B6 human genes
as well as for reference genes B2M and GADPH genes were obtained from Thermo Fisher: CYP3A4 (Hs00604506_m1), CYP2C9 (Hs02383631_s1), CYP2B6 (Hs04183483_g1), GAPDH (Hs02758991_g1), and B2M (Hs00984230_m1).
3
CYP2C9 Activity Assay with POR Variants
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The activity of CYP2C9 promoted by WT or mutant POR was tested using the fluorogenic substrate BOMCC (Invitrogen Corp, Carlsbad, CA, USA). The purified CYP2C9 (CYPEX, Dundee, Scotland, United Kingdom) was used to test the activities of the POR variants using 20 µM BOMCC as substrate. In vitro CYP2C9 assays were performed using a reconstituted liposome system consisting of WT/mutant POR, CYP2C9, and cytochrome b5 at a ratio of 5:1:1. The final assay mixture consisted of 5 µg DLPC (1,2-Dilauroyl-sn-glycero-3-phosphocholine) and proteins (1 µM POR: 200 nM CYP2C9: 200 nM b5), 3 mM MgCl2, 20 µM BOMCC in 100 mM Tris-HCl buffer PH 7.4, and the reaction volume was 100 µL. The P450 reaction was started by addition of NADPH to a final concentration of 1 mM, and fluorescence was measured on a Spectramax M2e plate reader (Molecular Devices, Sunnyvale, CA, USA) at an excitation wavelength of 415 nm and an emission wavelength of 460 nm for BOMCC.
4
CYP2C9 Activity Assay with POR Variants
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The activity of CYP2C9 promoted by WT or mutant POR was tested using the fluorogenic substrate BOMCC (Invitrogen Corp, Carlsbad, CA, United States). The purified CYP2C9 (CYPEX, Dundee, Scotland, United Kingdom) was used to test the activities of the POR variants using 20 µM BOMCC as substrate. In vitro CYP2C9 assays were performed using a reconstituted liposome system consisting of WT/mutant POR, CYP2C9 and cytochrome b5 at a ratio of 5:1:1. The final assay mixture consisted of 5 µg DLPC (1,2-Dilauroyl-sn-glycero-3-phosphocholine) and proteins (1 µM POR: 200 nM CYP2C9: 200 nM b5), 3 mM MgCl2, 20 µM BOMCC in 100 mM Tris-HCl buffer pH 7.4 and the reaction volume was 100 µL. The P450 reaction was started by addition of NADPH to a final concentration of 1 mM, and fluorescence was measured on a Spectramax M2e plate reader (Molecular Devices, Sunnyvale, CA, United States) at an excitation wavelength of 415 nm and an emission wavelength of 460 nm for BOMCC.
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