Frequently Asked Questions - Subcellular Fractions
Can I measure phase II metabolism using microsomes or S9 fractions?
Yes, but with microsomes and S9 you must add the necessary phase II cofactors. For example, if you want to measure glucuronidation (UDPGT) in a microsomal incubation, you will need to add uridinediphosphoglucuronic acid (UDPGA) in addition to the normally required NADPH regenerating system. As an alternative, consider using cryopreserved hepatocytes, since these contain all of the cofactors necessary to perform coupled phase I/II metabolism.
Can I repeatedly thaw and refreeze microsomes and S9 if I only need to remove a small aliquot of these products from a vial?
Yes, as long as you carefully thaw these products on ice, remove what you need, and then quickly refreeze the vial to -70° C. As an alternative to repeated thawing and freezing, you can thaw the vial once and distribute the contents into single-use vials for future use. That way, you only have to thaw the microsomes/S9 one more time.
How many animals are used to make each lot of microsomes and S9?
The number of animal livers used to make a batch of microsomes or S9 varies, depending on the species. For small animals such as mice and rats, we typically use at least 25 livers to make each lot of product. For larger animals such as monkeys and dogs the number is much smaller, typically 3 to 7 livers per lot of product.