Hi Ashraf,

Our favorite long used protocol was the following one. Male Wistar rats (5–6 weeks of age) were used to prepare the liver S9 fraction. The S9 consisting of both microsomal and cytosolic fractions, was obtained by centrifugation of whole-liver homogenate at 9000 × g for 20 min. The S9 fraction was further centrifuged at 105,000 × g for 60 min to separate the microsomes and cytosolic fraction. The incubation mixture consisted of the indicated concentrations of substrate of interest (usually 0.1 mM) in 5 μl ethanol, an NADPH-generating system (0.5 mM NADP+, 5 mM glucose-6-phosphate, 5 mM MgCl2), and 50 mg liver equivalent of S9 fraction or microsomes and/or cytosolic fraction in a final volume of 1 ml 100 mM potassium phosphate buffer (pH 7.4) containing 0.1 mM EDTA. For the inhibition study of P450, 0.1 mM SKF 525-A was also added to the reaction mixture. The incubation was carried out at 37°C for 60 min with shaking. After addition of 1 ml of 20% trichloroacetic acid (TCA), the quenched reaction mixture was allowed to stand for 15 min on ice, then was centrifuged at 2500 rpm for 10 min. The incubation system without an NADPH-generating system, or inactivated by addition of TCA prior to incubation, was used as the control. A 1.8-ml aliquot of the resultant supernatant was passed through a Sep-Pak Plus C18 cartridge  preconditioned with 10 ml methanol and 20 ml water for solid-phase extraction. The cartridge was washed with 10 ml water and the remaining water was purged by flushing with nitrogen gas. The adsorbed substances were eluted with 3 ml ethanol, and the eluate was evaporated to dryness in vacuo. The residue was dissolved in 200 μl ethanol, then 10 μl of the sample solution was transferred to a well of a microtiter plate for monooxygenase activities.

Best wishes,

Ilya 

Thanks Ilya for this detailed and comprehensive reply, Just asking:

1. is the  9000 × g centrifugation done at room tempersture or with cooling?

2. Which type of homogenizer you used, glass-teflon for example or other types?

3. What is the workup if I want to detect metabolites and % conversion by HPLC or LC-MS?

Again, thank you

Creative Bioarray is staffed with a group of well-experienced experts in drug metabolism and is able to provide customized S9 stability assays to assess the metabolic stability of your candidate drugs (microsomal stability assay is also available upon request).

https://www.creative-bioarray.com/Services/S9-Stability-Assay.htm