| Assay Method Information | |
| | Inhibition of Cytochrome P450 Enzymes in Vitro |
| Description: | Table 3: Experimental methods and procedures1) Prepare test compound and standard inhibitor working solution (100×);2) Take the microsomes out of the refrigerator at -80℃, thaw them on ice, put a date label, and put them back in the refrigerator immediately after use;3) Add 20 microliters of matrix solution to the corresponding wells;4) Add 20 microliters of phosphate buffered saline solution to the blank well;5) Add 2 microliters of test compound and positive control working solution to the corresponding wells;6) Add 2 microliters of solvent to inhibitor-free wells and blank wells;7) Preparation of working fluid of human liver microsomes;8) Add 158 microliters of HLM working solution to all the wells of the incubation plate;9) Preheat the board in a 37°C water bath for about 10 minutes;10) Prepare NADPH cofactor solution;11) Add 20 microliters of NADPH cofactor to all incubation wells;12) Mix and incubate all CYPs for 10 minutes at 37°C in a water bath;13) At the time point, stop the reaction by adding 400 microliters of cold solution (200ng/mL tolbutamide in acetonitrile and 200ng/mL Labetalol in acetonitrile);14) The sample was centrifuged at 4000 rpm for 20 minutes to precipitate the protein;15) Transfer 200 microliters of supernatant into 100 microliters of high performance liquid chromatography water, and shake it for 10 minutes;16) Start LC/MS/MS analysis. |
| Affinity data for this assay | |
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