Both mouse liver and fecal samples are suitable for this assay, as researchers have demonstrated the effectiveness of the kit for these types of samples.
Reference: Han, W., Zhang, D., Zhang, P. et al. Danlou Recipe promotes cholesterol efflux in macrophages RAW264.7 and reverses cholesterol transport in mice with hyperlipidemia induced by P407. BMC Complement Med Ther 23, 445 (2023). https://doi.org/10.1186/s12906-023-04253-9
Before homogenizing liver samples, ensure that all blood and body fluids are removed. It is also important to perform an initial serial dilution to determine the optimal amount of sample to use for testing.
Fecal Sample Preparation Protocol:
Add 2 ml of absolute ethanol to 200 mg of the stool sample in a tube.
Sonicate the mixture in a bath sonicator for 30 minutes.
After sonication, place the tubes in a heating block and reflux at 100°C for 15 minutes.
Centrifuge the tubes at 1500xg for 10 minutes, then transfer the supernatant into new tubes.
Resuspend the pellet in 2 ml of 80% ethanol, then repeat the reflux and centrifugation steps.
Combine the supernatants from this step with the first supernatants.
Resuspend the pellet a third time in 2 ml of chloroform/methanol (1:1, v/v), and again reflux and centrifuge.
Pool the supernatants once more.
Wash the pellet by adding 2 ml of chloroform/methanol (1:1, v/v), then centrifuge and remove the supernatant as before.
Dry the combined supernatant using nitrogen gas, a desiccator, or a vacuum pump.
Store the dried supernatant frozen at -20°C for later assays or, if using it immediately, resuspend it in 200-500 µL of water. Use the resuspended supernatant in the assay.
The kit operates based upon metabolism of a bile acid by 3–hydroxysteroid dehydrogenase. Any bile acid that can be metabolized with 3-alpha HSD will be detected in the assay.
