V3000
Jugular Vein or Carotid Artery Catheterization
Jugular Vein or Carotid Artery Catheterization (please specify in your order).
$75.90 (external)
V3001
Cannulation of cerebral ventricle
Implantation of a cerebral ventricle cannula allows investigators to evaluate physiological responses following central administration of various compounds. Anesthetized mice are placed in a digital stereotaxic apparatus (0.001 mm accuracy, Cartesian Instruments) specifically designed for mice. The dorsal scalp will be shaved, wiped with a betadine solution, and then a small midline incision over the dorsal surface is made to allow access to the cranium. After the affixed centering scope is used to "zero" lambda and bregma landmarks, a single guide cannula (2.5 mm length, 26-gauge, Plastics One) is positioned 1.0 mm above the lateral ventricle (coordinates: 0.6 mm posterior to bregma, 1.5 mm lateral to midline, 1.4 mm below the surface of the skull) and fixed to the skull using two stainless steel screws and dental cement. The incision in the scalp is then closed with surgical thread. Animals are removed to a post-surgical warming bed, and then individually housed for several hours until fully awake. Animals will be allowed to recover from surgery for a minimum of 7 days prior to testing, during which time a 30-gauge dummy cannula is left inside the guide cannula to prevent blockage.
$92/per mouse
V3002
Jugular vein and carotid artery catheterization
Arterial catheterization allows investigators to sample arterial blood as required for adequate glucose clamping (Niswender et al. J. Biol. Chem, 1997, Halseth et al. Am. J. Physiol. 1999) or other infusion/sampling purposes (Rottman et al. Am. J. Physiol. 1999) Catheterization of the right jugular vein allows the infusion of hormones, substrates, and tracers into the systemic circulation. The jugular venous catheter can be used to sample venous blood in long-term experiments because the jugular vein catheter will work for almost a month.
Arterial catheters are made from polyethylene tubing (PE-10) that is connected to silicone tubing (0.3 mm I.D., and 0.64 mm O.D.), 25 mm long. Jugular vein catheters are made from silicone tubing (0.3 mm I.D., and 0.64 mm O.D.). These catheters are connected to stainless steel tubes (0.3 mm I.D., 0.41 mm O.D., 15 mm) bent into an L shape. On the free end of the L shaped stainless steel tube a 20 mm piece of micro-renathane tubing (0.36 mm I.D., and 0.84 mm O.D.) is attached. The L shaped stainless steel tubes, attached to an arterial and a jugular vein catheter, are bundled together with silicone tubing (0.76 mm I.D. and 1.65 mm O.D.) and anchored with silastic medical adhesive (Silicone Type A). The catheters and the micro-renathane-stainless steel tubing will be heat sterilized.
The mouse is anesthetized and its skin on the interscapula and ventral surface of the neck is depilated by plucking. The depilated area is sterilized with 10% povidone-iodine. A small longitudinal incision (about 5 mm) is made in the skin over where the anterior jugular, acromeodeltoid, and cephalic veins join together. The connective tissues surrounding this junction are carefully removed. Two thin threads of silk (6-0 Silk, Davis+Gech) are passed under the jugular vein below the level of the junction. They are separated by about 3 mm. The cephalic thread, placed just below the joint, is tied to prevent bleeding. A small incision is then made just below the ligature, and the catheter is pushed 13 mm into the lumen. The catheter is fixed with the second thread and the thread previously used to tie the jugular vein. The common carotid artery is separated from the vagus nerve and muscle, and then two thin threads of silk (6-0 Silk, Davis+Gech) are passed under the artery. The cephalic thread is tied to prevent bleeding and then the artery is clamped by small bulldog clamp. A small incision is made just below the ligature, and the catheter is inserted into the lumen. The clamp is taken off and the catheter is pushed in 10 mm. The catheter is fixed with a second thread and the thread previously used to prevent bleeding. A blunt needle (16 gauge) is carefully inserted through the incision on the interscapula and pushed subcutaneously until the end comes out through the incision in the neck. The catheters will be carefully seized and pulled slowly through the needle. The incisions in the skin are then sutured. The catheters are connected to the stainless steel tubes. The bent portion of the stainless steel tubing is implanted under the skin and the incision is sutured. The implanted catheter is flushed with saline containing 200 U heparin/ml and 1 mg ampicillin/ml. Then the micro-renathane tubing is closed with a stainless steel wire. The mouse is injected subcutaneously with 150 mg/kg ampicillin. The total duration of the operation is about 50 min. Animals are removed to a post-surgical warming bed, and monitored until fully awake. Postoperative body weight and food intake are measured daily.
$136.40
V3003
Glucose Tolerance Test (Oral, i.p., Intravenous, or gastric catheter)
Oral glucose tolerance tests are performed on conscious mice with catheters chronically implanted directly in the stomach and the carotid artery. Intravenous glucose tolerance tests are performed on conscious mice with catheters chronically implanted in the jugular vein and carotid artery. Glucose will be given at 1g/kg or 2g/kg. These doses lead to peak blood glucose levels of 250 mg/dl to 400 mg/dl in wild type C57/bl/6 mice.
oral/ip: $36.30; With catheters: consult with Director
V3004
Glucose turnover
A primed (2 µCi) continuous infusion of [3-3H]glucose (0.4 µCi/min) is used to assess the rates of glucose appearance (Ra) and disappearance (Rd). Tracer is infused to allow a steady state to be reached then blood samples are taken to assess arterial glucose specific activity. Ra will be estimated as the ratio of the rate of infusion of [3-3H]glucose and the steady state plasma [3H] glucose specific activity (dpm/mg). Under steady state conditions, the rate of glucose disappearance (Rd) equals the rate of glucose appearance. The rate of glucose clearance is calculated by dividing the Rd by the arterial glucose concentration. Application of this technique is described by Niswender et al. J. Biol. Chem. 1997, and She et al. Mol. Cell. Biol. 2000.
Consult with Director - included with V3005
V3005
Hyperinsulinemic clamp
The hyperinsulinemic clamp is used to measure insulin action in vivo. Hyperinsulinemic clamps are performed on conscious mice with catheters chronically implanted in the jugular vein and carotid artery. A continuous infusion of insulin is given. Glucose levels are monitored in arterial samples every 5-10 min using an Accuchek glucose analyzer that allows the analysis of glucose with only 1 µl of blood. Glucose is infused in the jugular vein catheter at rates necessary to achieve the desired glucose level, based on feedback from arterial glucose measurements. These methods allow assessment of the responsiveness of the body to insulin. Blood from a donor animal is infused to maintain blood volume. By combining this technique with the tracer method one can also examine the impact of insulin on suppression of endogenous glucose production. (1, 2)
1. Ayala JE, Bracy DP, Malabanan C, James FD, Ansari T, Fueger PT, et al. Hyperinsulinemic-euglycemic Clamps in Conscious, Unrestrained Mice. J Vis Exp. 2011:e3188.
2. Halseth AE, Bracy DP, Wasserman DH. Overexpression of hexokinase II increases insulin and exercise-stimulated muscle glucose uptake in vivo. Am J Physiol. 1999 1/1999;276(1 Pt 1):E70-E7.
$639/mouse with [3-3H] glucose + [14C] deoxyglucose (includes surgery and analysis and hormones)
V3006
Hyperglycemic clamp
The responsiveness of the pancreas to glucose is assessed using the hyperglycemic clamp. Hyperglycemic clamps are performed on conscious mice with catheters chronically implanted in the jugular vein and carotid artery. Our clamps include 11 arterial insulin measurements and 3 arterial c-peptide measures.
A defined hyperglycemic stimulus is created using a primed variable glucose infusion to raise the glucose level to twice basal for 120 min. An established priming algorithm is used to elevate glucose quickly.
Mice with extra copies of the glucokinase gene locus were demonstrated to have a blunted insulin response to hyperglycemia using this technique (Niswender et al. J. Biol. Chem. 1997).
$598 (includes surgery, insulin + c-peptide measurements and analysis)
V3007
Gluconeogenesis & glycogenolysis (from hepatic 14C-UDPglucose and PEP)
The contribution of gluconeogenesis to the rate of glucose appearance is estimated from the specific activities of 14C-labeled hepatic uridine diphosphoglucose (this is assumed to reflect the specific activity of hepatic glucose 6-phosphate), and hepatic phosphoenolpyruvate (PEP) following the infusion of [U-14C]lactate. [3-3H]glucose is used to measure the rate of glucose appearance. Gluconeogenesis is equal to the rate of glucose appearance x [14C]UDP-glucose specific activity/[14C]PEP specific activity x 2. Glycogenolysis is equal to the difference between rates of glucose appearance and gluconeogenesis. The contribution of the kidney to these measurements is assumed to be small.
Consult with Director
V3008
Glycogen synthesis
Using [U-14C]glucose, the incorporation of the carbon of glucose into glycogen can be measured. If the ratio of [14C]UDP-glucose to blood glucose specific activity is assessed the fraction of glycogen formation from direct and indirect pathways can be calculated.
Consult with Director
V3009
Amino acid kinetics
The turnover of phenyalanine (3H ring 2,3,4,5,6 phenylalanine) , glutamine (U-14C-glutamine) and leucine (1-14C-leucine) is assessed by a primed continuous infusion of their respective isotopes for 2 hours (0.2-0.4 µCi/min). Blood samples (20 µl) are taken after a steady state is reached to assess plasma amino acid specific activity. Blood samples are mixed with an equal volume of 6% sulfosalicylic acid. Incorporation of tracer in tissue protein is used to assess tissue specific protein synthesis.
Consult with Director
V3010
Tissue specific glucose uptake
Tissue specific glucose uptake is assessed by measuring the tissue specific uptake of [2- 3H]-deoxyglucose([2-3H]DG). [2-3H]DG is infused (0.2 µCi/min) for 40 minutes or injected (12 µCi) . Arterial plasma samples are taken to determine the time course of [2-3H]DG during the 40 min period. [2-3H]DG is transported into cells and phosphorylated to yield [2-3H]DG-6-phosphate which is trapped in muscle. After 40 min mice are anesthetized with an intravenous infusion of pentobarbital and tissues of interest are rapidly removed and frozen in liquid nitrogen. This method has been applied during insulin- and exercise-stimulated conditions (Halseth et al. Am. J. Physiol. 1999).
Consult with Director
V3011
Tissue specific fatty acid uptake
Tissue fatty acid uptake is assessed by measuring tissue-specific incorporation of circulating 125I-BMIPP (Rottman et al. Am. J. Physiol. 2002). The beta-methyl modification of the long-chain fatty acid BMIPP (beta-methyl-p-iodophenylpentadecanoic acid) causes terminal trapping in the TCA cycle. Studies in man and a variety of small animal models, including rodents, have shown that BMIPP uptake and metabolism closely tracks that of endogenous long-chain fatty acids in a variety of normal and pathophysiologic states.
BMIPP is dissolved in proprionic acid, and incorporation of 125I is catalyzed with CuSO4. After extraction, the purified 125I-BMIPP is dissolved in ursodeoxycholic acid, filtered, and adjusted to defined activity. This stable compound is suitable for direct intravascular injection. Serum levels are stable in tracer amounts after injection, and tissue incorporation is measured by gamma counting of freeze-clamped samples in protocols compatible with the simultaneous assay of, for example, [2-3H]DG.
Consult with Director
V3012
Indirect calorimetry / energy expenditure in the Promethion
Whole body VO2 and VCO2 is measured continuously in conscious mice using a Promethion system (Sable Systems Int). The system is sensitive enough to measure small changes in VO2, VCO2 and RQ. They can be used to measure resting or exercising (running wheels or treadmill) gas exchange and energy expenditure. The Promethion is very advanced, allowing for measurement and control of food or water intake. In the Promethion the animals are housed in regular home cages with normal bedding. Activity is measured using beam breaks and converted to pedestrian locomotion. Food and water intake are very accurately quantified. The system integrates all of these data to monitor behaviors and patterns. Body weight and composition is included and measured before and after the metabolic cages.
All mice remain in the Promethion cages for 5 days with continuous data collection, allowing for acclimation while also providing extensive data. Full data is reported to the investigator in excel format.
$96.80
V3013
Exercise capacity/Exercise Stress Test (metabolic response to exercise)
Exercise is an integrated measure of fitness. Abnormal exercise capacity and decreased activity are a hallmark of most severe cardiovascular and metabolic diseases, and changes in exercise capacity are sensitive and early markers of cardiac and metabolic dysfunction. Thus abnormalities can be revealed with exercise that may not otherwise be manifested. Gas exchange techniques can be used during treadmill exercise in the mouse to describe the metabolic cost of exercise. Substrate fluxes and metabolism can be assessed isotopically during exercise in chronically catheterized mice (Halseth et al. Am. J. Physiol. 1999).
Treadmill exercise can be used to quantify the capacity of a mouse for either endurance or high intensity exercise. Peak exercise capacity and VO2 max will be measured using a closed gas exchange treadmill. Acclimated mice will exercise will at 10 m/min, 0° grade, increased to 14 m/min, 3 minutes later and then increased by 4 m/min a every 3 min thereafter up to 46 m/min or until mouse is exhausted. Exhaustion is defined as the mouse sitting on the shock pad and unable to get off. Time to exhaustion and speed at exhaustion is recorded.
$61.60
V3014
Spontaneous exercise activity
Spontaneous exercise activity is measured using a recording wheel placed in the cage during a 48 h period. The light dark cycle will be stringently controlled to minimize diurnal variations and training effects will be minimized by placing an identical wheel in the cage for the 24 hrs preceding the test measurement. Variables recorded include total distance traveled, peak speed and exercise duration.
Consult with Director - included with V3012
V3015
Food Consumption
Food consumption is assessed using an automated feeding apparatus that continually measure feeding behavior in an unobtrusive manner by allowing animals free access to food cups that are mounted on balances. The apparatus currently is capable of measuring and time-stamping individual weights from 16 balances simultaneously every 30 seconds and downloading the data directly to a computer for subsequent analysis. Therefore, cumulative food consumed and the time at which feeding bouts occur are continuously monitored. All feeding studies are done after the animal has acclimatized to the facility for at least 24 hours.
$4.20/day- included with V3012
V3016
Exploratory locomotor activity
Exploratory locomotor activity
Consult with Director- included with V3012
V3017
Assess real time imaging of cellular metabolic events
Assess real time imaging of cellular metabolic events will be done.
Consult with Director
V3018
In vivo optical imaging of gene expression
In vivo optical imaging of gene expression will be done.
Consult with Director
V3030
In vitro Morphology, Morphometrics and Histology (isolated heart)
A limited necropsy is conducted, noting gross observations, and removing and weighing the heart and lungs separately. After fixation, the heart is sectioned in a standard four-chamber view. Digital photographs on each heart is recorded and archived in a web-accessible format. Chamber and mural dimensions will be measured. The fixed hearts will be maintained in a physical archive, while one slice will be paraffin embedded. 4-5 sections will be cut and prepared for H& E and Masson trichrome. Digital photomicrographs will be recorded and archived together with summary evaluations. A cardiologist with special expertise in mouse cardiac development and histology reviews all gross and microscopic sections.
Consult with Director
V3031
Echocardiography, in vivo morphology, systolic and diastolic function; Stress echocardiography
Echocardiography is a non-invasive technique that can detect the presence of localized or generalized hypertrophy or thinning of the myocardium of the left ventricle (LV) and the presence of regional or global wall motion abnormalities associated with systolic dysfunction. The application of transmitral Doppler analysis allows the detection of abnormal filling patterns associated with LV diastolic dysfunction.
The core employs two echocardiography systems, one for high resolution imaging and a high throughput lower resolution imaging system. Echocardiography can be performed on conscious as well as anesthetized mice. In addition to imaging under basal conditions, the mice may undergo stress testing following the administration of dobutamine.
(1) Low-Resolution Echocardiography. A 15 MHz linear-array transducer (Sonos 5500, Agilent) is used for high throughput echocardiography in conscious mice. Measurements include LV wall thickness in the intraventricular septum and posterior wall, diastolic and systolic LV diameter, and heart rate. Fractional shortening and ejection fraction are computed as a measure of systolic function. This system also allows for transmitral Doppler analysis that can detect abnormal filling patterns associated with LV diastolic dysfunction.
(2) High-Resolution Echocardiography. The Vevo 2100 Imaging System (VisualSonics) utilizes a ultra-high frequency 18-38 MHz linear-array transducer with a digital ultrasound system for superior imaging of mouse cardiac morphology and function as well as Doppler analysis. Measurements include those described for the low-resolution echo. In addition, the VevoStrain analysis software provides a highly sensitive speckle-tracking based echocardiographic imaging technique that offers quantitation of the velocity of the walls, displacement, strain, strain rate, and time to peak analysis.
$27.60 low resolution; $72.00 high resolution using VEVO 2100
V3032
Telemetry (in vivo chronic arterial blood pressure measurement)
Because of difficulties in making physiological measurements in anesthetized mice, a commercially-available system for recording mean arterial blood pressure, heart rate, systolic, diastolic, and mean pressure are used. The primary probe is the PA-C10 which is completely implantable, reducing animal stress, and ensuring the most reliable data. Probes to measure ECG and blood glucose are also available.upon request. This service includes probe implantation, probe removal, data acquisition, and a partial probe refurbishment fee.
$593 per mouse for two week experiment.
V3033
Blood pressure measurements
Blood pressure (BP) represents an integrated measure of overall cardiovascular function, and is affected by stroke volume, heart rate, inotropic state, and vascular tone. Abnormalities of BP regulation (primarily hypertension, but also hypotension) are associated with major cardiovascular morbidity and mortality, and are epidemiologically associated with diabetes and obesity.
Three complementary technologies are available for the measurement of BP: 1) non-invasive tail-cuff plethysmography, 2) direct arterial measurement by intracarotid catheterization, and 3) telemetry via implanted catheter.
(1) Tail-cuff Plethysmography. Plethysmography (tail-cuff) is performed using a tail-cuff BP apparatus (BP-2000, Visitech Systems, Inc.). This technology is non-invasive and there is good concordance with the direct BP measurements described below.
(2) Carotid Catheterization. Direct arterial measurements are obtained via a chronically placed catheter in the carotid artery. The catheter is connected to a TXD-310 transducer and BP measured using a Digi-Med BPA 400 (Micromed). Experiments using this approach are typically coordinated with metabolic measurements.
A dual catheter approach (arterial and venous) allows for BP measurements in response to specific pharmacological infusions in the awake or anesthetized state; and enables evaluation of both peripheral and central mechanisms of BP regulation.
(3) Telemetry. Telemetered direct BP measurement is performed using an implanted micro-miniature device (PA-C10, DSI) implanted subcutaneously with the catheter typically placed in the right carotid artery. The mouse is housed individually in a cage placed over the receiver platform and BP data digitally recorded via the DATAquest A.R.T. system (DSI). Advantages of this approach include the ability to continuously record BP over a period of weeks, to assess the diurnal range in BP, and stress artifact induced by animal handling is avoided.
Tail-cuff Plethysmography-$8.40; Carotid Catheterization-$81.60; Carotid and jugular vein catheterization-$129.60; Telemetry-$593
V3034
Vascular morphology
A variety of tissues (heart, aorta, kidney, brain) can be processed in four µm sections. Aortic sections are examined for wall thickening, perivascular fibrosis, and fibrin deposition. The inner border, the lumen outer border, the tunica media are traced in each arterial image with Masson's trichrome stain and imaged at a magnification of 200X. The lumen ratio (the medial thickness to internal diameter and the area fibrosis (collagen deposition stained with aniline blue) surrounding blood vessels are calculated and compared. Perivascular fibrosis is determined as the ratio of the area of fibrosis surrounding the vessel wall to the total vessel area.
Consult with Director
V3036
Tail vein injections
Tail vein injections are done in the conscious mouse.
$7.20
V3050
Insulin
30 µl plasma for duplicate analysis. 5-day double antibody assay w/dilution procedure. Detection range: 0.01- 0.5 or 0.1-10 ng/ml. Interspecies or human specific assays are available.
$9.49/sample
V3051
Glucagon
60 µl plasma for samples with basal glucagon concentrations (~50 pg/ml). 30 µl plasma for concentrations >100 pg/ml. Duplicate analysis in plasma preserved in EDTA/Trasylol. 5-day double antibody assay. Detection range: 10-200 pg/ml.
$8.04/sample
V3052
Corticosterone
15 µl plasma for duplicate analysis. 1-day double antibody assay. Detection range: 25-1000 ng/ml.
$25.75/sample
V3053
Catecholamines
Epinephrine and Norepinephrine (HPLC)
50 µl plasma preserved in EGTA-glutathione 1-day assay. Detection range: 50-2000 pg/ml.
$10.75/sample
V3054
Leptin
Available as a Luminex assay
$16.33/sample
V3055
C-Peptide
Available as a Luminex assay
$38.56/sample
V3056
Growth Hormone (GH)
Available as Luminex assay.
$33.29/sample
V3058
TSH
TSH is assayed in a double-antibody RIA format. 55 micro liters of plasma is required for duplicate analysis. It involves a dilution step and is a 5-day double antibody procedure. The lower limit of the assay is 0.4ng/ml.
Consult with Director/Kit is available
V3059
PRL (prolactin)
Prolactin is assayed in a double-antibody RIA format. 25 micro liters of plasma is required for the assay. A dilution step is required and is 5-day double antibody assay. The lower limit of detection if 1.5ng/ml.
Consult with Director/Kit is available
V3060
ACTH
55 µl plasma for duplicate analysis. 5-day double antibody assay. Detection range: 5-100 pg/ml.
$37.25/sample
V3061
Insulin-like growth hormone-1 (IGF-1)
IGF-1 is a five-day double antibody procedure. 30 micro liters of plasma is used for extraction and duplicate analysis.
Consult with Director/Kit is available
V3062
Aldosterone
60 µl plasma for duplicate analysis. 2-day double antibody assay. Detection range: 5-100 pg/ml.
$19.57/sample
V3064
Resistin
Resistin may be analyzed in a double - antibody RIA format. It is a two day assay at room temperature. Sample volume: 50 µL Plasma, serum or tissue culture medium. The lower limit of detection is 0.78 ng/mL. Can also be run as single-plexed assays using luminex instrumentation. A dilution step is required for the adiponectin requiring a minimum of 5 ul of sample. The resistin does not require a dilution and may be multiplexed with other adipokines with a required sample volume of 25 ul for duplicate analysis.
Consult with Director/Kit is available
V3065
Adiponectin
Adiponectin may be analyzed in a double-antibody RIA format. It requires an overnight incubation at room temperature. Sample volume < 2 µL serum or plasma, or < 100 µL tissue culture media. A 1:400 x dilution is required. The lower limit of detection is 1.56 pg/mL. Can also be run as single-plexed assays using luminex instrumentation. A dilution step is required for the adiponectin requiring a minimum of 5 ul of sample. The resistin does not require a dilution and may be multiplexed with other adipokines with a required sample volume of 25 ul for duplicate analysis.
Consult with Director/Kit is available
V3066
Estradiol
35 µl plasma for duplicate analysis. 2-day double antibody assay. Detection range: 2-50 pg/ml.
$39.16/sample
V3067
Testosterone or Progesterone
25 µl (male) or 35 µl (female) plasma for duplicate analysis. 2-day double antibody assay. Detection range: 5-1000 pg/ml.
$15.61/sample
V3070
Plasma lipids
Enzymatic Free Fatty Acid Test Price - $15.60
Total plasma cholesterol and triglyceride are measured by standard enzymatic assays. HDL cholesterol is measured with the enzymatic method after precipitation of VLDL and LDL using dextran sulfate and Mg++. Using these data LDL cholesterol can be calculated using the Friedewald equation, if triglyceride levels are below 400 mg/dl. Investigators may request a total plasma lipid profile or specific plasma lipid measurements.
Free fatty acids are extracted from plasma using heptane/isopropanol. The heptane layer containing FFA is removed, plated on silica gel plates and developed in petroleum ether, ethyl ether, and acetic acid. The FFA band is scraped from the plate and FFA are eluted with heptane /isopropanol. The solvent is removed, and the FFAs are methylated. Methylated fatty acids are analyzed by gas chromatography. Depending on the assay a variety of chromatograph conditions and columns are utilized. A computer identifies each fatty acid peak and can provide data in a number of different ways including quantitation of mass of fatty acid, percent distribution of fatty acids present, quantitation of total lipid in the sample.
$9.60-Triglycerides; $9.60-Cholesterol; $9.60 HDL Cholesterol; $12.00 Enzymatic Free Fatty Acid Assay (FFA)
V3071
Lipid extraction, separation, quantification
Total plasma cholesterol and triglyceride are measured by standard enzymatic assays. HDL cholesterol is measured with the enzymatic method after precipitation of VLDL and LDL using dextran sulfate and Mg++. From these data LDL cholesterol is calculated using the Friedewald equation, as long as triglyceride levels are below 400 mg/dl. Investigators may request a total plasma lipid profile or specific plasma lipid measurements. Plasma FFAs are analyzed with a commercially available enzymatic kit from Wako Life Sciences. Free fatty acids are extracted from plasma using heptane/isopropanol and separated from other lipid classes by thin layer chromatography. The FFAs are methylated and analyzed by gas chromatography. Data: (1) total FFAs present (mg/ml plasma); (2) percent distribution of individual fatty acids in the FFA fraction. Tissue/Cell Lipid Analyses (Phospholipid, Diglycerides, Free Fatty Acids, Triglycerides, and Cholesterol Esters)
Lipids are extracted by the Folch-Lees method. Internal standards are added, and the lipid classes are separated by thin layer chromatography. The appropriate band(s) are scraped from the TLC plate, and the fatty acids from the lipid ester classes are methylated and analyzed by gas chromatography. Results: (1) total amount of lipid class present; (2) fatty acid profiles of the lipid classes presented as percent of total fatty acids. Total unesterified cholesterol is analyzed in the lipid extract by gas chromatography after addition of appropriate internal standard. An aliquot of the lipid extract is dried and saponified at 80 C in 1 N KOH in 90% methanol for 1 hour. The non-saponifiable sterol is extracted into hexane, concentrated under nitrogen, solubilized in carbon disulfide and analyzed by gas chromatography.
Lipoprotein fractions are separated from 100 microliters of plasma (or serum) by gel filtration column chromatography. Approximately 40 fractions (0.5 ml) are collected and the amount of triglyceride and cholesterol in each fraction is determined using microtiter plate, enzyme-based assays. Profiles of triglyceride and cholesterol are constructed. Calibration of the column with purified lipoprotein fractions permits quantitation of each lipid in various lipoprotein classes.
$14.40-Cholesterol; $14.40-Diglyceride; $14.40-Free Fatty Acid (FFA); $14.40- Phospholipid; $14.40-Triglyceride; $14.40-FFA by GC
V3072
Fatty acid profiles of lipid esters by gas liquid chromatography
Total lipids are extracted and lipid classes separated by TLC as described above. Lipid ester spots are scraped from the plates and methylated. Fatty acids of lipid esters can be methylated without removal of the lipid from the silica gel. However, in some applications, we have found it advantageous to elute the lipid from the silica gel prior to methylation. The fatty acid profile of the lipid class is also determined. By this method total lipid mass and fatty acid profile for each lipid is determined.
Consult with Director
V3073
Quantitation of individual phospholipid classes
Individual phospholipid classes are isolated by one dimensional TLC. A total lipid extract is applied to high performance TLC plates. To quantitate the individual classes, the spots are scraped from the plate, eluted and phosphorus is determined. To determine the fatty acid composition of the individual phospholipid classes, the spots are scraped from the plates and fatty acids methylated.
Consult with Director
V3074
Short chain fatty acid analysis by gas liquid chromatography
Plasma short chain fatty acids are analyzed by the following procedure: to 200 ul of EDTA plasma in a 1.5 ml Eppendorf microfuge tube is added 20 µl of internal standard and 1 ml of absolute ethanol. The sample is mixed thoroughly, centrifuged, and the supernatant is recovered. The sample is evaporated using a Speed Vac and dissolved in 15 µl water, and prior to injection 5 µl of orthophosphoric acid (25%) is added. The short chain fatty acids are separated on a 6' x 2 mm glass column packed with SP-1200/1%H3PO4 on 80/100 Chromosorb W AW.
Consult with Director
V3075
Fast Protein Liquid Chromatographic (FPLC) Lipoproteins Separations
Lipoprotein fractions are isolated using columns arranged in tandem to achieve complete resolution of the major lipoprotein classes from 1-2 ml of plasma. The columns are equilibrated in 50 mM phosphate-buffered saline and calibrated using lipoprotein fractions isolated by ultracentrifugation. Fractions (0.5 ml) are collected and the appropriate tubes containing the desired lipoprotein fraction(s) combined. The position of the major lipoprotein classes are determined by cholesterol (or triglyceride) assay on the column fractions using a microtiter plate enzyme-based assay. As an alternative method lipoproteins can be isolated by fast protein liquid chromatography.
This includes analysis of the composition of the fraction (protein and lipid) as well as morphologic analysis (sizing) by negative stain electron microscopy. For compositional analysis the lipoprotein fractions protein is analyzed using the BCA method with a modification to eliminate lipid interference. The samples are then lyophilized and delipidated using ethanol and ether. Lipid components are separated by TLC and analyzed by GLC and/or colorimetric assays.
$104.40/sample
V3076
Morphometric determinations (aorta)
Mice are sacrificed and flushed with 30 ml saline. The heart with ascending aorta is embedded and snap-frozen in liquid N2. Cryosections of 10 um thickness are taken from the region of the proximal aorta. Cryosections are stained with Oil Red O and counterstained with hematoxylin. In addition to the aortic cross-sections, whole aortas will be analyzed in "en face" preparations to evaluate the distribution and characteristics of atherosclerotic lesions in the distal aorta. After the removal of the heart and the aortic arch, the entire remainder of the thoracic and abdominal aorta is dissected from the carcass. An incision is then performed longitudinally and for the total length of the specimen, so to expose the inside face. The open face aorta is pinned out on a black cardboard submerged in saline, and then stained with Oil Red O.
Consult with Director
V3080
Gross examinations and necropsy
The standard necropsy procedure for diabetic mice includes an examination of the pancreas, heart, liver, eyes, peripheral nerves, peripheral vasculature, fat, and kidneys. Complete, intermediate, or limited necropsies will be performed with or without the aid of a dissecting microscope. Gross pathologic findings will be described, documented by digital photography and organs will be weighed.
Consult with Director
V3081
Tissue preparation, embedding, sectioning and routine staining
The default fixative will be 4% paraformaldehyde. This offers investigators the option of later performing in situ hybridization or laser microdissection and capture of protein, DNA or RNA from select populations. This fixative provides a minimal degree of cross-linking of the proteins, thereby rendering antigenic sites more accessible. Other fixatives are used as appropriate. If frozen sections are needed, tissues will be snap frozen in liquid nitrogen and sectioned on a cryostat. When molecular analyses are required, tissue samples can also be rapidly placed in Trizol or RNA extraction solutions and stored at -80°C until investigators retrieve their samples.
Consult with Director
V3082
Tissue microdissection
Potential applications of laser dissection include the selective microdissection of islet cells for gene expression studies, harvesting and analysis of specific glomerular cells in diabetic nephropathy, and harvesting endothelial cells from diseased microvasculature. This Core has available a PixCell II™ laser capture microdissection device (Arcturus Engineering) and a PALM microdissection scope. These workstations perform laser capture microdissection simply, quickly and precisely. They are capable of locating single cells or large groups of cells and, using a simple aim-and-shoot method to extract them for subsequent molecular analysis (DNA, RNA, or protein).
Consult with Director
V3083
Screen/optimize immunohistochemical protocols for mouse-specific commercial and custom-designed antisera
To stain mouse tissues with mouse monoclonal antisera we utilize Mouse-on-Mouse (MOM) kits (Vector Labs), Ark kits (DAKO Corp) or HistoMouse Kits. Although peroxidase-based protocols will be the mainstay of the Subcore with visualization by brown DAB or red AEC chromagens, staff is familiar with the alkaline phosphatase-based kit and its detection with the fuschin chromagen. Fluorescently-tagged primary, secondary, or tertiary antibodies in frozen sections, cultured cells, or for co-localization studies will be used as required to tailor protocols to address investigator needs. Non-fluorescent double immunostaining will also be performed in the Pathology Subcore using the Double Label Kit provided by DAKO. When prospective antisera fail to recognize antigenic sites in paraffin embedded sections or produce non-specific staining patterns, we advise investigators to proceed with frozen sectioning.
Consult with Director
V3090
Amino Acids - Full and Select Profiles (e.g. gluconeogenic) (HPLC)
50 µl plasma. Detection range: >1 µmol/l.
$38.22/sample
V3091
Specific selected amino acid profiles
50 µl plasma. Detection range: >1 µmol/l.
$30.71/sample
V3093
Specific activities for gluconeogenic and glycogenic assessment
UDP glucose, UDP -galactose and PEP specific activities in percholoric acid extracts of liver will be obtained by sequential chromatographic separations as described by Rossetti.
$94.80-UDPG; $91.92-PEP
V3094
Direct jugular vein injection and blood sampling
Direct jugular vein injection and blood sampling is done on conscious mouse.
$13.20
V3095
Myocardial infarction
Models of Cardiac Injury and Pressure Overload
Cardiovascular complications in metabolic diseases typically represent additive or synergistic effects of compound insults, i.e. phenotypic stresses (hypertension, myocardial infarction, etc.) and genetics. The ability to superimpose phenotypic stresses on transgenic or knockout mouse models allows for comprehensive and reproducible screening for cardiovascular complications and consequences of metabolic disease and diabetes in the mouse.
The core employs three widely used surgical models to induce cardiac stress or injury: 1) myocardial infarction, 2) myocardial ischemia-reperfusion injury, and 3) transverse aortic constriction.
Myocardial Infarction. The mouse myocardial infarction (MI) model is achieved by ligating a suture around the left main coronary artery of the LV thereby causing the infarction. The MI model is widely used for evaluating the response to myocardial injury or dysfunction. It also forms the basis for the majority of studies of myocardial repair, often obtained with stem cell injection. This model has been reliably implemented in the core with excellent survival, reproducible infarct size, and the ability to couple the infarction with long-term monitoring, stem cell injection, or metabolic studies. As an alternative to the coronary ligation method, an infarct can be induced by LV placement of a cryoprobe. This provides a smaller and more precisely sized infarct, with clear delineation of the border zone.
$72.00
V3096
Myocardial ischemia reperfusion
Models of Cardiac Injury and Pressure Overload
Cardiovascular complications in metabolic diseases typically represent additive or synergistic effects of compound insults, i.e. phenotypic stresses (hypertension, myocardial infarction, etc.) and genetics. The ability to superimpose phenotypic stresses on transgenic or knockout mouse models allows for comprehensive and reproducible screening for cardiovascular complications and consequences of metabolic disease and diabetes in the mouse.
The core employs three widely used surgical models to induce cardiac stress or injury: 1) myocardial infarction, 2) myocardial ischemia-reperfusion injury, and 3) transverse aortic constriction.
Myocardial Ischemia Reperfusion. Myocardial ischemia reperfusion injury is achieved in the same way as the MI model, except the suture is occluded for 10-15 minutes of ischemic time followed by release of the suture and reperfusion.
$60.00
V3097
Transverse aortic constriction
Models of Cardiac Injury and Pressure Overload
Cardiovascular complications in metabolic diseases typically represent additive or synergistic effects of compound insults, i.e. phenotypic stresses (hypertension, myocardial infarction, etc.) and genetics. The ability to superimpose phenotypic stresses on transgenic or knockout mouse models allows for comprehensive and reproducible screening for cardiovascular complications and consequences of metabolic disease and diabetes in the mouse.
The core employs three widely used surgical models to induce cardiac stress or injury: 1) myocardial infarction, 2) myocardial ischemia-reperfusion injury, and 3) transverse aortic constriction.
Transverse Aortic Constriction. Transverse aortic constriction (TAC) or aortic banding induces a pressure-overload on the heart and is achieved by placing a constriction around the transverse aorta. A successful TAC is confirmed by Doppler analysis of the flow velocity across the aortic constriction. Myocardial remodeling in the TAC mouse can be sequentially monitored by echocardiography as the heart progresses from the compensatory stage to decompensation and heart failure.
$72.00
V3098
GFR-FITC-Inulin; HPLC Cr
GFR will be measured in conscious mice based on the decay rate of plasma FITC-inulin following a single bolus intravenous injection of FITC-inulin. This method does not require urine collection, and GFR can be periodically measured in same mouse.
Additional approach for determining GFR in conscious mouse is based on creatinine clearance rate. Mouse 24-hour urine will be collected using metabolic cage. Plasma and urinary creatinine concentration will be determined using HPLC approach.
Consult with Director
V3099
Albuminuria
Mouse urinary albumin excretion rate can be determined by two methods: (1) measuring the albumin to creatinine ratio in spot urine sample; (2) measuring albumin concentration in urine collected over 24 hours using metabolic cage. Urinary albumin and creatinine concentration will be determined using cartridge-based DCA2000 (Bayer Diagnostics) or ELISA kits (Exocell Inc).
$15.60
V4000
Renal Blood Flow (Doppler)
Mouse renal cortical and medullary blood flow can be measured using a laser-doppler flowmeter (Tansonic Systems Inc). This system will also monitor blood pressure and heart rate. Renal function including urinary electrolyte excretion can be studied.
Consult with Director
V4001
Urine Na/K
Sodium and potassium concentration in mouse plasma and urine will be determined using an automatic flame photometer (Instrumentation laboratory Inc). This measurement requires 20 µl of plasma or urine.
Consult with Director
V4002
Osmometer Plasma/Urine
Plasma and urine osmolality will be determined using a freezing point osmometer (Precision System Osmette). This measurement requires 50 µl of plasma or urine.
Consult with Director
V4003
Urine Ca/Phosphorus Excretion
Calcium and phosphorus are two important electrolytes in the urine. The concentration of urinary calcium and phosphorus will be determined using colorimetric assay (BioAssay Systems). The measurement for calcium and phosphorus requires 5 µl and 50 µl of urine, respectively.
Consult with Director
V4004
Urine pH
The pH can be determined in as little as 5 µl of urine (or other body fluid) using a Mini Combo pH Electrode (World Precision Instruments).
Consult with Director
V4005
Glycemic Control using Minimed
Blood glucose levels over 72 hours can be monitored in conscious mice using Medtronic MiniMed CGMS System (Medtronic). In this system, blood glucose level is determined based on glucose concentration in interstitial fluid. A correlation between glucose levels in the blood and interstitial fluid in mice has been previous demonstrated. A fiber probe will be implanted subcutaneously. This probe will detect interstitial glucose levels every ten seconds over three days and the signals will be stored in a glucose monitor. The probe will be removed after the experiment and the mice can be sent back to the researcher.
Consult with Director
V4006
Personnel Training
Personnel Training (experimental or surgical training)
Consult with Director
V4007
Surgical Training
Surgical training for a variety of surgery techniques (catheterization, telemetry, bariatric surgery, implants)
$58.30 (external)
V4010
Ghrelin - Active (RIA)
30-60 µl plasma for duplicate analysis in HCL/PMSF. 5-day double antibody assay. Detection range: 5-100 pg/ml.
$35.36/sample
V4011
Ghrelin - Total (RIA)
30-60 µl plasma for duplicate analysis. 5-day double antibody assay. Detection range: 100-1000 pg/ml.
$35.36/sample
V4012
Glucose (Enzymatic)
40 µl of plasma for duplicate analysis. Detection range: 20-900 mg/dl.
$5.41/sample
V4013
Progesterone (RIA)
35 µl plasma for duplicate analysis. 2-day double antibody assay. Detection range: 10-5000 pg/ml.
$15.61/sample
V4014
Purine Nucleotides (HPLC)
In situ collected and weighed, 100 mg of liver (liquid nitrogen), add 1 ml cold 0.4M perchloric acid with 0.5M EGTA, homogenize, sit 10 min on ice, spin ~3200g@4C for 10 min, decant supernatant, add 8 drops 0.5M K2CO3, mix, sit 5 min on ice, spin ~3200g@4C for 5 min, decant, save supernatant, freeze / analyze.
$79.82/sample
V4015
T3 (RIA)
30-60 µl plasma for duplicate analysis. 5-day double antibody assay. Detection range: 0.05-5 ng/ml.
$36.26/sample
V4016
T4 (RIA)
30 µl plasma for duplicate analysis. 5-day double antibody assay. Detection range: 0.5-20 ng/ml.
$36.27/samples
V4017
Luminex Assays
Mouse Single Plex Adiponectin
Mouse Cytokine/Chemokine Panel
Mouse Metabolic Hormone
Mouse Pituitary
Mouse/Rat IGF
Mouse Th17
These are most frequently used assays. For more information, go to http://hormone.mc.vanderbilt.edu/prep.vmsr or http://hormone.mc.vanderbilt.edu/price.vmsr
First plex $38.56; Additional plex $7.20; Luminex use fee (per plate) $73.92
V4018
Acetaminophen
Acetaminophen
$32.21
V4019
Cortisol - Plasma
Cortisol - Plasma
$9.04/per sample
V4020
Cortisol - Salivary
Cortisol - Salivary
$12.14/per sample
V4021
Creatinine
Creatinine
$139.39/per sample
V4022
PYY - Active
PYY - Active
$17.20
V4024
Bariatric Surgery (Roux-en-Y, VSG, biliopancreatic diversion, sham controls)
Bariatric surgery (Roux-en-Y gastric bypass, vertical sleeve gastrectomy, biopancreatic diversion, and appropriate sham controls) . The modified gastric bypass in the mouse: In this procedure the stomach is bypassed and the food flows through the bypass arm directly into the jejunum.
The biliopancreatic diversion procedure in the mouse: This procedure is historically thought to be a malabsorptive procedure and has several variations in the human. In brief, the biliary and pancreatic secretions are physically separated from gastrointestinal chyme flow until a point near the terminal small bowel. In theory this leads to significant malabsorption, though when this procedure is done clinically many times a gastric restriction component is also added.
RYGB $155; VSG $46.20; Biliary Diversion $47.30; Shams $46.20
V4026
Body Composition Analysis by NMR
Body composition analysis by NMR analysis (Bruker Minispec). This measurement does not require anesthesia of the mouse. Body composition will provide lean mass, fat mass, total body weight, adiposity (%) and % lean mass for each mouse.
External: $18.70/mouse. Internal (VU/VUMC): $8.00/mouse (self-use) or $17.00 (w/assistance)
V4033
PYY - Total
PYY- Total
$17.20/per sample
@NationalMMPC
Mouse Phenotyping