At or below -70°C?
Based on our experience, microsomes are stable indefinitely at or below -70°C. Refer to the manuscript by Pearce et al., 1996 for data establishing the stability of XenoTech's microsomes.

CAUTION: The cited study was performed by XenoTech's personnel using our proprietary processes, and the stability data only applies to microsomes prepared by XenoTech.

On ice?
Microsomes are stable on ice, 2-8°C, for several hours; however, such exposure should be minimized, consistent with the experimental design. Microsomes should not be left on ice, or refrigerated, overnight. Microsomes that are thawed and maintained on ice for less than 2 hours can be re-frozen at -80°C and reused without significant loss of enzyme activity.

At room temperature?
Microsomes are typically thawed in a room temperature water bath. For a 0.5-mL vial of microsomes, this procedure typically takes 2 to 5 minutes. After the microsomes are thawed, they should be immediately placed on ice until used. Exposure to room temperature should be minimized as much as possible depending on the experimental design

At 37°C?
Microsomes are typically incubated at 37°C for determination of enzyme activity. Stability of microsomes at 37°C should be ascertained based on the enzyme activity of the metabolic reaction being studied. This is achieved by verifying that product formation is directly proportional to incubation time at one-half and at twice the normal/desired incubation time. XenoTech does not recommend incubating microsomes at 37°C for more than 2 hours. If extended incubations are carried out, experiments should be performed to verify that the enzyme activity is stable during the incubation period. Microsomes that have been incubated at 37°C should not be reused or refrozen.

Microsomes should be stored at -80°C until needed. Exposure to higher temperatures, such as
2-8°C (on ice), room temperature, or 37°C, should be minimized as much as possible and as allowed by the experimental design. The important thing to remember is that the rate of enzyme degradation is proportional to the increase in temperature of the microsomes.

Our recommendation: thaw the microsomes quickly in a room temperature water bath, and immediately place them on wet ice. Subsequently, the microsomes may be diluted or added to incubations directly. Vortex the microsomes as recommended below to assure homogeneity. The diluted microsomes or incubation mixtures are kept on ice until you are ready to start your incubations. Typically, incubations are carried out at 37°C.

XenoTech's pooled human liver microsomes are carefully designed to yield final activities that represent the "average" in a bank of human liver microsomes.

We first characterize the individual samples of liver microsomes with respect to CYP enzyme activities and exclude any samples that have unusually high or low activities. We then determine the theoretical average of the proposed pool of microsomes and compare it with the average activities from a large bank (>500) of characterized human livers. When the values meet specifications, the pool is prepared by blending samples from up to 50 individual donors, with each donor being significantly represented in the pool.

All pools are made in large batches, making these products available for an extended period.

Yes: gender-specific pools of microsomes, S9 fraction and cytosol are available from human, monkey, dog, rat and other species.  Check our Products section for more details.

Yes, microsomes are in suspension and should be well-mixed to obtain a homogeneous sample. XenoTech’s microsomes are stable under agitation caused by short, 2- to 10-second periods of vortexing. In fact, we recommend that the microsomes be vortexed each time they are to be diluted, and immediately before starting incubations.

Unlike rats, where there are enormous sex differences in CYP enzyme activities, there are insignificant gender differences in human CYP enzyme activities.

For more information, please see our poster on the topic: The Effects of Gender, Age and Ethnicity on Human Cytochrome P450 Activity

Pearce R, McIntyre CJ, Madan A, Sanzgiri U, Draper AJ, Bullock P, Cook DC, Burton LA, Latham J, Nevins C and Parkinson A: Effects of freezing, thawing, and storing human liver microsomes on cytochrome P450. Arch Biochem Biophys, 331: 145-169, 1996.

Robertson P, DeCory H, Madan A and Parkinson A: In vitro inhibition and induction of human hepatic cytochrome P450 enzymes by modafinil. Drug Metab and Dispos, 28: 664-671, 2000.

The properties of the CYP enzymes do not vary from one ethnic group to the next. However, CYP levels and the incidence of genetic polymorphisms vary among ethnic groups. In other words, there are quantitative but not qualitative differences in CYP enzymes.

For more information, see our poster on the topic: The Effects of Gender, Age and Ethnicity on Human Cytochrome P450 Activity

Dosing varies for each treatment group; this information is included on the data sheet that accompanies your order.  

Results of serum tests for known pathogens (HIV, Hepatitis B and C, CMV) are included with your order. XenoTech does not sell subcellular fractions from donors known to harbor infectious diseases, such as HIV, hepatitis or syphilis. However, we recommend that all human products be handled as potential biohazards and universal precautions be followed.

XenoTech's microsomes are suspended in 250 mM sucrose. You may use 250 mM sucrose to dilute the microsomes or you may use a buffered solution, such as potassium phosphate buffer (typically pH 7.4). If you choose the latter, remember to account for the concentration of phosphate buffer when calculating the final concentration of phosphate ions in the incubation mixture.

The pools are engineered to yield results representative of an overall population and to minimize lot-to-lot variation, however, the data will differ somewhat.

XenoTech prepares large batches of microsomes for most products to minimize changes in lot number.  If you have a specific project in mind, or are planning for annual usage, we'll be happy to prepare and characterize a custom lot for your company.

1. XenoTech’s scientists have been preparing and using microsomes for P450 research since the early 1980s.
2. We have performed careful analysis and published data regarding the freeze-thaw stability and < –70°C stability of our microsomes.
3. Because microsomes serve as the test system for a variety of studies, the microsomes are characterized with GLP principles in mind.
4. We use LC/MS/MS methods to characterize the enzymatic activities in our microsomes for greater precision and reliable data.  Substrates are chosen based on FDA recommendations and clinical relevance, and we include multiple substrates for CYP3A4.
5. We use our microsomes for in-house contract studies, which allows us to appreciate and anticipate the needs of our customers.  This practice also provides an ongoing quality check for the same products we offer for sale.
6. By producing large batches of products, we make it easier for end users to complete their research with the same unique lot of product.  This reduces the need to validate more than one test system during the course of their study.

Each lot of our microsomes (human or animal, untreated or induced) is characterized with respect to cytochrome b5 content, cytochrome P450 content, NADPH-cytochrome c reductase activity and at least one CYP enzyme activity. The activity of each of ten CYP enzymes, FMO and five UGTs is determined for each lot of pooled human liver microsomes. These data are compiled into lot-specific data sheets, which accompany your order.

Certain demographics are available on all human donors, including sex, age, race, limited medical history (major illnesses, recent medications, etc.) and limited social history (smoking, alcohol, drug habits, etc.). Information that may reveal the identity of the donor is not available.

Perform a carbon monoxide binding difference spectrum as published by XenoTech scientists.* If you see a large peak at 420 nm, it is likely that P450 has started to degrade to its inactive form of P420.

Caution: hemoglobin, often found in the microsomes as a contaminating protein, can give a peak at 420 nm also. Follow the Matsubara method described in the publication referenced below.

Alternatively, you can perform an enzyme assay and compare the rates you measure with those provided on the product datasheet.

*Pearce R, McIntyre CJ, Madan A, Sanzgiri U, Draper AJ, Bullock P, Cook DC, Burton LA, Latham J, Nevins C and Parkinson A
(1996) Effects of freezing, thawing, and storing human liver microsomes on cytochrome P450. Arch Biochem Biophys 331:145-169.

Yes, please contact us to help you find the corresponding subcellular fractions. Availability may be limited.

Microsomes are derived from S9 by a centrifugation process which separates the microsomal fraction of S9 from the cytosol, concentrating the CYP enzymes in the microsomal pellet. As a consequence, CYP activities in the microsomal fraction are approximately four to five times greater than that of its parent S9 fraction.