Metabolic and Analytic Core

The Luminex 200 Instrument with open-architecture xMAP® Technology enables multiplexing of biological assays to quantify protein expression levels in various biological samples. Systems using xMAP® Technology perform discrete assays on the surface of color-coded microspheres read in a compact analyzer. Using multiple lasers or LEDs and high-speed digital-signal processors, the analyzer reads multiplex assay results by reporting the reactions occurring on each individual microsphere particle.

Assessments are performed following established guidelines. For the glucose tolerance test, overnight fasted animals have their glucose assessed prior to dosing (oral, intraperitoneal, intravenous) and whole blood samples are taken at 15-30-minute intervals for 120 minutes. A commercially available glucose meter is used to assess glucose levels. Insulin tolerance tests are performed in the same manner as the glucose tolerance test but with a 6-hour fasting time, and whole blood collected prior to insulin dosing. Whole blood is collected every 15-30 minutes for 120 minutes and glucose levels are assessed.

Lipoprint analyses: LDL and HDL sub-fraction distributions can be efficiently analyzed and quantified with the Quantimetrix Lipoprint system (Quantimetrix Corporation, Redondo Beach, CA). This relatively simple benchtop system, originally purchased with NIH INBRE funds (P20GM103466), utilizes high resolution polyacrylamide gel electrophoresis to separate and measure the amount of cholesterol in each LDL and HDL sub-fraction. The Lipoprint, which was developed based on this report, allows for the rapid quantification of lipoproteins fractions with only 25μl of fasting plasma samples (not previously frozen). Up to 7 LDL bands can be detected and measured. The LDL-1 and -2 bands correspond to large particles, whereas bands LDL-3 to -7 correspond to small dense LDL particles. Similarly, 10 HDL sub-fractions can be detected. HDL-1 to -3 represent large particles, sub-fractions -4 to -7 are reported as medium particles, and sub-fractions -8 to -10 as small particles. This system uses a proprietary software to calculate each lipoprotein fraction according to a total cholesterol value obtained for each plasma sample. LDL and HDL kits for the analysis of 100 samples (per kit) are available.

Whole-body energy balance in live mice and rats is assessed via indirect calorimetry using the Panlab OxyletPro® System (Harvard Apparatus, Barcelona, Spain). Adult animals are weighed and individually housed in metabolic cages. Following a 24-hr acclimation period, food and water intake, physical activity, and energy expenditure (O2 consumption, CO2 production) are measured over a 24-48-hr period. Data collection and calculations are performed using the Panlab METABOLISM software.

To evaluate systemic lipid uptake, mice will be subjected to an oral lipid challenge. Briefly, adult animals are fasted for 14hrs overnight in standard cages with access to water ad libitum. The animals are weighed, and gavaged with 0.5 mL of olive oil at t=0. Blood samples are collected from the tail at t=0, 30, 60, 90, 120, 180, and 240 min. The amount of TG present in blood plasma at each time point is determined using a Wako L-Type TG-H kit (Richmond, VA, USA).

The Seahorse XF96 Analyzer is the gold standard platform in metabolic assays today and able to assess substrate utilization, mitochondrial function, metabolic switching, and cellular health. The Seahorse XF96 provides fast and sensitive measurements of cellular bioenergetics without labels in a microplate format, which enables time-resolved analysis and reuse of the cells. The probes consist of fiber optic wave guides affixed to systematically dispersed sensors to monitor hydrogen ions to give the extracellular acidification rate (ECAR), which is an indicator of glycolysis and oxygen to give the oxygen conception rate (OCR), an indicator of respiration. Through application of different substrates and/or inhibitors glycolysis, mitochondrial dysfunction, oxygen consumption, and fatty acid oxidation can be assessed.

The Multi-electrode array (MEA) system can be readily used for electrophysiological studies to understand electrically active cells such as neural networks and cardiac syncytium. MEAs capture the field potential or activity across an entire population of cells. With far greater data points per well, it is capable of detecting activity patterns that would otherwise elude traditional assays such as patch clamp electrophysiology. MEA systems provide a state-of-the-art solution to high-throughput in vitro electrophysiology.

The i-STAT System is a point-of-care-testing platform that utilizes single-use i-STAT test cartridges offering a broad menu of tests on a single, portable platform. Each test cartridge has a unique combination of biosensors facilitating a wide range of diagnostic tests, including blood gas, blood chemistry and electrolyte measurements.

Blood Pressure (BP) is the pressure (force per unit area) exerted by circulating blood on the walls of blood vessels, and constitutes one of the principal vital signs. The pressure of the circulating blood decreases as it moves away from the heart through arteries and capillaries, and toward the heart through veins. Blood pressure is measured using a cuff sensor on the tail while the mouse is restrained on a warming platform.

An echocardiogram, often referred to in the medical community as a cardiac ECHO or simply an ECHO, is a sonogram of the heart. Also known as a cardiac ultrasound, it uses standard ultrasound techniques to image two-dimensional slices of the heart. In addition to creating two-dimensional pictures of the cardiovascular system, an echocardiogram can also produce accurate assessment of the velocity of blood and cardiac tissue at any arbitrary point using pulsed or continuous wave Doppler ultrasound. This allows assessment of cardiac valve areas and function, any abnormal communications between the left and right side of the heart, any leaking of blood through the valves (valvular regurgitation), and calculation of the cardiac output as well as the ejection fraction.

To assess the functional capacity of the animals, a series of treadmill-based exercise tolerance tests may be performed on a motor-driven treadmill (Exer 3/6 Treadmill, Columbus Instruments, Columbus OH, USA) following a 5-day acclimatization period in which animals will be familiarized with treadmill running for 5 minutes/day at speed of 5 m/min up a 5% incline. Endurance capacity and critical speed (CS), the asymptote of the speed to duration relationship, will be determined in each animal using a protocol described previously [Ferguson, S.K., et al., J. Applied Physiology, 2020. 129(3)]. Mice will undergo a series of five runs to exhaustion at speeds of 10, 20, 30, and 40 m/min performed at random on separate days for the determination of critical speed (CS).

With the addition of an IVIS Lumina XRMS in vivo imaging system, which offers cutting edge in vivo fluorescence and bioluminescence technology combined with low dose 2D X-ray, we can use imaging as a fast and powerful technique to non-invasively study molecular and biological processes of disease. The IVIS Lumina XMRS System allows for non-invasive and precise metabolic analyses and integrates optical signals in the anatomical context of rodents and larger animals. This gives new expanded capacity to accommodate up to 3 animals simultaneously and allows for longitudinal, disease progression studies.

The DSI Small Animal Telemetry System allows for data acquisition from conscious, freely moving laboratory animals, providing stress-free data collection while enhancing animal welfare. Its implants are offered in various sizes to support a range of research models, including mice, rats, dogs and non-human primates. In addition, this system is the only one that allows for continuous blood pressure and glucose monitoring in mice, an option not available in any other system at JABSOM.

The Aurora 3-in-1 Whole Animal System provides flexible and accurate measurement of rodent muscle properties in-situ, in-vivo and in-vitro. The system gives researchers a simple method to test mechanical properties of skeletal muscle. By combining in-situ, in-vivo and in-vitro muscle tests using one simple platform, researchers can capture a complete picture of muscle physiology in rodent subjects. Our test system with murine apparatus is complete with temperature-controlled animal and limb plates designed to support and fix the animal and limb being tested. The system can be converted to an isolated (in-vitro) muscle test system with the attachment of an optional 25mL bath. Of note, muscle samples are attached at only one point to measure force and length, saving time and increasing productivity.

To assess the effects of in vivo acute exercise on metabolism, a treadmill running exercise protocol can be performed on a motor-driven treadmill (Exer 3/6 Treadmill, Columbus Instruments, Columbus OH, USA). Following determination of maximal running speed, for the actual exercise treatment, the mice will be randomly assigned to a rest or exercise group, and exercised at a relative workload of ~65% of their maximal running speed at a 10˚ incline for 20 minutes. Control mice of the “rest” group will be placed on a still treadmill for 20 minutes. The experimenter will be blinded as to the genotype of the mice.

One of the most commonly used surgical intervention for pressure-overload induced hypertrophy is coarction of the ascending aorta i.e. aortic banding. This system has been very well characterized and proven to be highly reproducible with a low mortality rate of 10-20% or less in experienced hands. Aortic banding is an excellent model system to evaluate the process of development of left ventricular hypertrophy in response to hemodynamic stress. Furthermore, after several months, a subset of animals progresses into heart failure.

Two surgical methods of left ventricular infarction are commonly done in the Phenotyping Core; cryo-ablation and left anterior descending artery (LAD) ligation. Cryo-ablation is a procedure in which a portion of left ventricle is frozen with a liquid nitrogen cooled probe, resulting in tissue necrosis. See next topic for description of LAD ligation.

(LAD) ligation is a procedure to ligate the left anterior descending artery to induce an infarction. In addition, a LAD ligation can be performed as a component of ischemia-reperfusion studies, where the ligature is removed after a set period of time.

A pump based on a piston powered by osmotic pressure which pumps drug/s into the body over an extended period. The unit is implanted under the skin and has no external or protruding parts.

If you have a procedure in mind that is not listed here, please do not hesitate to let us know. We are well versed in many procedures that are not necessarily listed here.

We use a catheter-based system for tail vein injections allowing for continual infusions (when used in conjunction with a pump). We also perform traditional direct syringe/needle tail vein injections used for single bolus delivery.

Using one of several available mouse strains that are susceptible to atherosclerosis such as apolipoprotein E-null (ApoE-/-) or low- density lipoprotein receptor-null (Ldlr-/-) mice, studies involving the feeding of high fat and high cholesterol diet can be performed to induce atherosclerosis in mice. The appropriateRequested tissues can be harvested at the end of the study and the extent of atherosclerosis can be quantitatively measured.

The principal purpose of the cardiovascular system is the delivery of oxygen to other organs of the body. A convenient and noninvasive way to perturb this function is to expose mice to a hypoxic environment. We offer chronic exposure to hypoxia in the MPC as it can also serve as a valuable stress to reveal subtle phenotypes of cardiovascular limitation in any mouse model. We can combine the wheel running capability described above with the hypoxia chamber to evaluate limitations on exercise capacity.

Training and advice for general mouse metabolic testing and surgery techniques. Please contact us for details.

The Diabetes Research Center’s (DRC) Metabolic and Analytic Core (MAC) is sponsored by the National Institutes of Health, National Institute of General Medical Sciences as a resource to provide services to the community of scientists who use in vitro and in vivo models to study diabetes, obesity, diabetic complications, and other metabolic diseases. In order to accomplish this goal, the AMC offers to researchers consultation and phenotyping services that require specialized expertise or equipment. Modest fees for these tests are set at actual cost. A purpose of the Diabetes Research Center’s Metabolic and Analytic Core (Core) is to acquire, maintain, and operate instruments critical to research related to health sciences. The Core provides access to these instruments on a cost-recovery basis. This document states the expectations for usage of the Diabetes Research Center’s Metabolic and Analytic Core. Users and their supervisors must read and agree to the policies contained in this document before gaining access to the instrumentation within the facility.

1.1. All users must undertake an initial mandatory training on any instrument they are planning to operate without Core support.

1.2. Users must submit an online Request to request training. Training is held during normal business hours only (Monday to Friday, 8 am to 4 pm).

1.3. Following the initial or successive training sessions, it will be determined by Core personnel whether the user is capable of competently operating the instrument independently. A user’s first 5 hours of independent use of an instrument must be scheduled during normal business hours and Core personnel must be notified by email so that they may be accessible to the user.

1.4. During the first training session, users must submit a signed user agreement.

2.1. Independent access to the instruments is restricted to users who have demonstrated competency using the instruments. Independent after-hour access is restricted to those who have 5 or more hours of notified use during normal business hours, are currently enrolled students and employees of the University of Hawaii or employees of the Research Corporation of the University of Hawaii, and possess key cards with programmed access to the rooms and/or buildings in which in the instruments are housed. Exceptions can be granted by the directors of the DRC and the Core.

2.2. If there is a long lapse without using equipment (3 months or more), the Core reserves the right to revoke after-hours independent access, pending demonstration of proficiency. For lapses of 6 month or more, a refresher or full re-training may be required, pending evaluation by Core personnel.

2.3. Users without key card access can utilize the instrumentation during business hours under the supervision of Core personnel. Exceptions can be granted by the directors of the DRC and the Core. Assisted sessions and training take place during normal business hours only.

2.4. Access to the IVIS Lumina imaging instrument in the Biosciences Building Vivarium is limited to users who are trained and approved for access to the Vivarium. The Animal and Veterinary Services (AVS) can provide information regarding specific requirements for access to the Vivarium (Michael Wong, D.V.M., wongmich@hawaii.edu; Lisa Sato, lisaho@hawaii.edu).

2.5. In order to access instrument computers, users must have an individual computer login that is provided upon training. Shared use of login information is strictly prohibited and may result in termination of access to the Core.

2.6. Users are required to sign in on the paper log sheets next to each instrument. The requested information in these log sheets helps to track any problems with the instruments as well as usage of lamp time, gasses, and other consumables. All users must keep track of their time on all instruments or software, whether the user logs in to the computer or not and whether there is an associated fee or not.

2.7. There is no training offered for metabolic flux measurements (Seahorse XFe96), which is only provided as a service performed by trained personnel depending on instrument availability.

3.1. The Core has a first come, first served policy regarding instrument reservation and providing services.

3.2. All users must schedule personnel time or reserve instrument time by submitting an online Request.

3.3. All reservations must be honored, or cancelled, with a minimum of 24 hours advance notice. For cancellation of reservations done with less than 24 hours notice, Core personnel must be notified immediately. There should be justification for a short-notice cancellation to avoid charges. Failure to cancel reservations is a violation of Core policies.

3.4. Users who are late for their scheduled appointment (30-min grace period) may lose the booked time.

3.5. Users must adhere to the schedule, and vacate the equipment promptly at the end of the booked time if another user is scheduled to use the instrument.

3.6. Repetitive violations of these instrument reservation policies may result in suspension of user privileges, pending retraining and a new user agreement.

4.1. All users must be in compliance with laboratory and biosafety training requirements established by the University of Hawaii Environmental Health and Safety Office (EHSO) www.hawaii.edu/ehso/training/ and the Office of Research Compliance Biological Safety Program (BSP) https://research.hawaii.edu/orc/biological-safety/.

4.2. Use of any hazardous material that may be biological and/or chemical, must be reviewed and received prior approval by the Core director. The user is obligated to provide SOPs, which must include handling of the sample and control of potential spills and decontamination. Radioactive material or samples requiring BSL-3 or BSL-4 conditions cannot be handled under any circumstances in this facility.

4.3. For any samples, manipulation should be kept to a minimum within the facility. It is highly recommended that sample preparation (changing media, pipetting, mixing, etc.) be performed in the user’s lab before the samples are transferred to the facility. All biological and chemical waste must be disposed of in the user’s lab.

4.4. For live-cell manipulation, all materials (media, etc.) should be prepared in the user’s lab and must be fully contained (e.g. closed perfusion system). All biological and chemical waste must be disposed of in the user’s lab.

4.5. During any active manipulation of hazardous materials in the facility, users must wear the appropriate PPE, which needs to be provided by the user’s PI. Any spills, sprays or other potential contamination must be handled immediately and reported to Core personnel and JABSOM’s Environmental Health and Safety Office.

5.1. Users must cooperate with Core personnel to ensure that the instrumentation is maintained in optimal conditions.

5.2. Any indication of malfunctioning of an instrument must be immediately reported to Core personnel and recorded on the log sheet(s).

5.3. Users must never attempt to repair or modify the instruments. The facility is responsible for the cost of repairs and calibrations. Cost for repairs only becomes the responsibility of the user’s supervisor if the damage is due to: (a) an untrained person operating the instruments without Core personnel supervision, or (b) evidence of user’s negligence.

6.1 Storage on Core Computers
All data must be saved in the designated user’s network drive, flash media, or external hard drive. Any data left on the computer may be deleted at the end of the day without warning. In the event of a problem storing data, Core personnel must be notified so the data can be temporarily saved to Core drives. Downloading anything from the internet onto Core computers is strictly prohibited.

6.2 Data Ownership
All data generated from a submitted strain belongs to the submitting investigator and his/her institution. Diabetes Research Center personnel have no rights to use this data for personal or institutional research purposes unless a formal, documented arrangement of collaboration exists between DRC personnel and the investigator. The NIH strongly encourages the sharing of research data. NIH guidelines regarding data sharing can be found at http://grants1.nih.gov/grants/guide/notice-files/NOT-OD-03-032.html.

There is no charge for instrument training up to five hours, nor for use of software. Following training, the instruments listed under the Equipment tab can be used independently at the rates shown. If users would like Core personnel to run their samples, or are in need of extended technical assistance on instruments, Core personnel can assist at the rates shown on the Rates tab. Time of use is based on records generated through log sheet records. An account number must be furnished for invoicing prior to using the instruments. The PI or supervisor charged will receive an invoice from the Diabetes Research Center in the month following the service period.

The existence of core facilities depends in part on acknowledgment in publications and grants, and it enables the core to obtain financial support so essential services can be provided in the most economical way possible. Therefore, please acknowledge the Metabolic and Analytic Core in all peer-reviewed publications and grant applications in which data was obtained using Core equipment, expertise, or services; and acknowledge the National Institutes of Health Center for Biomedical Research Excellence Program: P20GM113134 NIGMS.