Consultation with MPC Staff is required before using any service for the 1st time.

Please allow 1-2 months’ notice to allow sufficient time to amend ACURF protocols and plan your experiments appropriately. Contact MPC Staff for the appropriate ACURF documents and applications.

Mitochondrial Bioenergetics: Seahorse XFe24 and O2K from OROBOROS

Seahorse XFe24

The XF-24 analyzer simultaneously measures the Oxygen Consumption Rate (OCR) and the Extracellular Acidification Rate (ECAR) of cultured cells, isolated mitochondria, islets or small tissue samples.  The instrument measures mitochondrial function in intact cells utilizing real-time bioenergetics - the rate of aerobic respiration and lactic acid production via glycolysis, respectively.  The system performs label-free, non-destructive measurements, which allow the cultured cells to be used for further assays.

The plate accommodates 24 individual wells, and up to 4 different test compounds can be injected separately into each well.  A Cell Mito Stress Test typically injects oligomycin, FCCP, and rotenone/antimycin-A.  A Glycolytic Rate Assay typically injects rotenone/antimycin-A followed by 2-deoxy-glucose.

This service includes consultation with the Core director for proper experiment design, and a first time explanation of data produced.  First time users should contact the Core director at least 2 weeks before running an experiment.  Users will provide the Core with their cell plate on the day of the experiment.  The Core will provide all materials needed to run a standard assay.  XF assay takes at least 4 hours. Data will be provided by the next day.

The Seahorse Bioscience website has a wealth of information. It is highly recommended that users research their area of interest before beginning an experiment.

O2K from Oroboros

The O2K performs high resolution respirometry, perfect for small tissue samples (permeabilized or not) and isolated mitochondria.  The   Core owns two systems with two chambers each allowing for four simultaneous measurements to increase workflow.  There are many experimental designs possible depending on each investigators needs.  Example: Buffer -> Tissue Sample -> ADP -> Substrate such as Pyruvate/Malate -> Rotenone (Complex I inhibitor) -> Succinate (Substrate for Complex II) -> Oligomycin (ATP Synthase Inhibitor) -> FCCP (Uncoupler)

This service includes consultation with the core director and/or consultant for proper experiment design, and a first time explanation of data produced.  First time users should contact the Core director at least 2 weeks before running an experiment.  We have space available for preparation of samples for ease of running the experiment (for a nominal fee), or users can prepare samples in their own labs and simply come to the Core when ready to use the O2K.

NMR (Nuclear Magnetic Resonance)

We have a Bruker Minispec available to measure whole body composition in mice without anesthetics.  The LF50 is designed specifically for mice up to 60-70 grams. Investigators will receive analysis of fat tissue, lean tissue, and free fluid.  They are relatively simple systems to use.  First time users must be trained by Core personnel.  Thereafter, they can use the machine on their own.

GTT/ITT

A glucose tolerance test is commonly used in estimating the overall insulin sensitivity in vivo in rodent models. The glucose/insulin will be given intraperitoneally (by injection, IPGTT) after a fast period to deprive food in the GI track. Blood samples are taken at intervals (0, 5, 15, 30, 60, 90 minutes post injection) to determine how quickly the glucose is cleared from the blood as well as the corresponding changes of insulin in plasma. 

Metabolic Chambers (Indirect Calorimetry):  Promethion (Sable Systems International) or CLAMS (Columbus Instruments)

Metabolic Chambers determine whole animal energy expenditure. This is a non-invasive measurement of food intake, energy expenditure, respiratory exchange ratio and physical activity.  Promethion and CLAMS have many similarities and some differences.  The chart below depicts some of these differences.

Promethion Data (Sable Systems International)
The Core will provide investigators with a data analysis for Promethion that includes many parameters, averaged for light and dark cycles.  The Promethion system records absolute raw data and can be analyzed in many ways.  What the Core provides is a basic analysis.  Any interest in analyzing data in a different manner should be discussed with the Core director.

CLAMS Data (Comprehensive Lab Animal Monitoring System, Columbus Instruments)
The Core will provide investigators with a data analysis for CLAMS including HEART, RER, ACTIVITY, SLEEP, and FEED, averaged for light and dark cycles.  If you implant the radiotelemeters you can simultaneously calculate core body temperature.  Investigators must take special consideration in planning to use radiotelemeters as it requires surgery. 

Clamps

Clamps assess whole body insulin action and are considered to be the “gold standard” method to assess insulin sensitivity.

Investigators should know we suggest an N=6-8+ per group.  The fail rate is ~20% (usually due to clotted catheter, lost catheter, or surgical complication).

Hyperinsulinemic Euglycemic Clamp

• This procedure investigates glucose metabolism and its sensitivity to insulin. The typical standard for clamps in mice is to study in the conscious state using a chronic venous catheter.
• Basic clamp: determines solely whole-body insulin sensitivity.
• Detailed clamp: Tissue specific insulin action can be determined using glucose tracers: 3-3H-glucose (liver) and/or 14C-2-deoxyglucose (muscle, fat, etc). Two doses of insulin are often studied, as organs have differential sensitivity. Lipid and hormonal responses are often measured.
• The procedure timeline includes surgical catheter placement with anesthesia, 5-10 days postoperative recuperation, then after 4-5 hour fast when the actual clamp is performed followed by euthanasia.  If radioactive tracers are used there are post-clamp assays performed that can take 2-4 weeks.

Hyperglycemic Clamp

• This procedure investigates insulin secretion in response to controlled, standardized hyperglycemia.
• The procedure timeline includes surgical catheter placement with anesthesia, 5-10 days postoperative recuperation, overnight fast, 2 hour acclimatization, and then 2 hours of controlled hyperglycemia followed by euthanasia.