Bile Acid Assay Kit

Both equations will provide the same value. In both cases, (Fsample-Fblank) is multiplied by 20 and by n, and divided by (Fstandard-Fsample). It does not matter in what order this is performed.

The kit does not need to be used all at once. There is no specific threshold for freeze/thaw cycles to avoid, but it is advisable to limit these cycles. Aliquoting reagents is recommended if the intention is to use the kit for four or more cycles.

If this product has an expiration or retest date, it will be shown on the Certificate of Analysis (COA, CofA). If there is no retest or expiration date listed on the product's COA, we do not have suitable stability data to determine a shelf life. For these products, the only date on the COA will be the release date; a retest, expiration, or use-by-date will not be displayed.
For all products, we recommend handling per defined conditions as printed in our product literature and website product descriptions. We recommend that products should be routinely inspected by customers to ensure they perform as expected.
For products without retest or expiration dates, our standard warranty of 1 year from the date of shipment is applicable.
For more information, please refer to the Product Dating Information document: https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/marketing/global/documents/418/501/product-dating-information-06-25-mk.pdf

The concentration of the prepared internal standard in step 1 is 80 µM. Since the ratio of the internal standard to sample volume is 1:4, dividing 80 µM by 4 yields 20 µM, which represents the effective concentration of the internal standard.

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.

Products may be shipped at a different temperature than the recommended long-term storage temperature. If the product quality is sensitive to short-term exposure to conditions other than the recommended long-term storage, it will be shipped on wet or dry-ice. If the product quality is NOT affected by short-term exposure to conditions other than the recommended long-term storage, it will be shipped at ambient temperature. As shipping routes are configured for minimum transit times, shipping at ambient temperature helps control shipping costs for our customers. For more information, please refer to the Storage and Transport Conditions document: https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/marketing/global/documents/316/622/storage-transport-conditions-mk.pdf

Unfortunately, the kit has not been validated with cell culture supernatant, and there are no relevant citations for this particular use. While the expected level of bile acid in HepG2 cell culture supernatant is unknown, given that significant amounts of unconjugated bile acids are released into the medium of HepG2 cell cultures, the sample might be compatible with the kit. It is advisable to conduct an initial serial dilution of the sample to identify the appropriate dilution factor that yields results within the linear detection range (1-150 uM bile acid). The Sample Blank should be adjusted to accommodate possible interference in the sample matrix. If the initial results are not satisfactory, further dilution of the sample may be necessary.

The sample volume required for Kit MAK309 is optimized as per the specifications provided in the kit bulletin. Diluting the plasma 20X is not advisable as it would result in a loss of assay sensitivity. Upon confirming with the vendor, it was advised that the sample volume can be scaled down to half the volume in each step, with the other components adjusted accordingly. However, diluting the samples is not recommended as it would compromise sensitivity. Alternatively, the assay can be adapted for use in a 384-well plate or a half-area 96-well plate, such as CLS3880, which have smaller well volumes and can accommodate smaller sample volumes. For further details, refer to the Scaling Down Volume Procedure from the Vendor document.

A black, round-bottom plate can be used for this assay instead of a black, clear-bottom plate. Although there may be slight variations in the standard curve (resulting in a higher slope compared to a black flat-bottom plate), the standard curve remains linear, making the black round-bottom plate suitable for use.

The MAK309 assay has not been tested in a NaOH solution. It is believed that it should work, but if the NaOH solution is basic, it may interfere with the enzymes in the kit. Neutralizing the pH of the NaOH solution before performing the assay is recommended if possible. Additionally, a test run of the kit could be conducted by preparing the internal standard in NaOH. It is important to use NaOH as the blank instead of water to adjust for any changes in the measured bile acid concentration due to the use of NaOH.