Project protocol — Contents
Workflow and sampling
Equipment and supplies
Reagents and solutions
Procedure for plasma lipids assays
Procedure for body composition determination using NMR
Step Procedure performed Equipment Data collected 1 Mice fasted for 16 h - - 2 Blood samples obtained by retroorbital bleed under isoflurane anesthesia blood collection kit - 3 Blood samples mixed with heparin anticoagulant and processed to obtained plasma for subsequent assays centrifuge - 4 Plasma samples prepared and assayed for lipids automated workstation and plate reader plasma total cholesterol (CHOL), unesterified CHOL, HDL, LDL plus VLDL, FFA, TG 5 Mice measured for body composition NMR percent body fat
- Beckman TJ-6 (or comparable) centrifuge
- Beckman Biomek 2000 Automated Workstation
- 96 well plate reader Molecular Devices Spectramax-Plusmicroplate reader
- 0.65 mL minitubes (Phenix #M-931B)
- 96 well round bottom plates (USA Scientific #5665-0101)
- 96 well flat bottom plates (USA Scientific #5665-5101)
- 37°C water bath
- Nuclear magnetic resonance (NMR) using Bruker Minispec
- Software from Echo Medical Systems (Houston, TX)
- Heparin anticoagulant
- 0.9 % sodium chloride solution
- Reagents for triglyceride and triglyceride blank assays: Triglyceride reagent (Sigma #F6428 triglyceride [free glycerol] reagent) reconstituted as per manufacturers recommendations and 8000 U lipase (EMD Biosciences #437707). Reagent for triglyceride 'blank' assay is made exactly the same way but the lipase is omitted.
- Glucose assay reagent (Fisher #SB1070-125)
- Glucose standard (Sigma #G6152)
- Triglyceride standard (Sigma #G556-100mL)
- Cholesterol standard (Fisher #SB1012-030)
- FFA assay reagents (reconstituted per manufacturers recommendations)
- NEFA Color reagent A (Wako #999-34691)
- NEFA Solvent A: (Wako #995-34791)
- NEFA Color reagent B: (Wako #991-34891)
- NEFA Solvent B: (Wako #995-35191)
- NEFA Standard solution (1 mEq/L) (Wako #276-76491)
- Control 1- SER-T-FY-1 Level 1 human control serum (Stanbio #G427-86), (Boerne, TX, USA)
- Control 2 is SER-T-FY-2 Level 2 human control serum (Stanbio #G428-86), (Boerne, TX, USA)
- Cholesterol Assay Reagent:
- 4-amino antipyrine (Aldrich #A39300)
- KCl (Fisher #P217-500)
- sulfonic acid (Research Organics #6062H-3)
- sodium cholate (USB #13630)
- PIPES (Sigma #P-3768)
- Triton X-100 (Sigma #T-6878)
- horseradish peroxidase (Amresco #0417)
- cholesterol oxidase (EMD Biosciences #228250)
- cholesterol esterase (EMD Biosciences #228180) [Omit the cholesterol esterase from the reagent for unesterified cholesterol (UC) assay]
- Heparin/MnCl2 for precipitation in HDL assay
- MnCl2 (Fisher #M87-100)
- Heparin solution (EMD Biosciences #375095)
a. Mice are fasted for at least 16 h.
b. Mice are bled retro-orbitally under isoflurane anesthesia, 3-6 h after the beginning of the light cycle.
c. Whole blood is collected with heparin anti-coagulant.
d. Blood samples are processed to separate plasma for lipid assays.
e. For total plasma cholesterol, samples are treated with cholesterol esterase to hydrolyze cholesterol esters, such that all of the cholesterol ester and unesterified cholesterol pools are measured.
f. For unesterified cholesterol, plasma sample is not treated with cholesterol esterase, such that only the free or unesterified cholesterol is determined.
g. Unesterified cholesterol value is then be subtracted from the total cholesterol to calculate the esterfied or cholesterol esters (CE).
h. For HDL cholesterol, plasma sample is treated with Heparin/MCl2 to precipitate ApoB-containing lipoproteins before performing the lipid assay.
i. For triglyceride assay, the mass of glycerol released after the hydrolysis of fatty acids from triglycerides is determined.
j. A triglyceride (glycerol) "blank" assay is conducted to measure the endogenous levels of glycerol present in the plasma prior to the hydrolysis of triglycerides.
k. This endogenous "blank" value is then subtracted from the total glycerol determined after the hydrolysis of triglycerides in order to correct for the endogenous plasma free glycerol concentrations, which are considerably higher in mice than in humans.
Internal quality control
a. All assays are run in triplicate assays on a 96 well plate using Molecular Devices SpectraMax plus plate reader.
b. Standards with known analyte concentrations are run on each plate as well as control samples in order to validate the accuracy of the assay.
c. Unknown samples (26, in triplicate) are conducted on each plate, with the remainder of the wells used by 3 standards, 2 control samples, and 1 "blank" sample run in triplicate. Thus, a total of 32 minitubes are labeled for each assay.
External quality control
a. Participate in the Centers for Disease Control and Prevention Lipid Standardization program (laboratory ID # is LSP-25).
b. Receive 12 test samples quarterly from the CDC, and weekly run of unknown samples includes one of the CDC test samples.
c. To be notified of meeting CDC criteria for accuracy and precision, the values obtained from the test samples are submitted at the end of each quarter.
Investigator's note: "We have passed each quarter for the past 18 years."
Preparation of Plasma Samples, Standards, Controls, and Blanks
Dilution of plasma samples
a. Frozen mouse plasma samples (~100 µL/tube) are thawed on ice.
b. Samples are vortexed and centrifuged briefly in a table top microcentrifuge to recover all of the plasma.
c. Depending on the lipemic state, the plasma is diluted from 4 to 16 fold with 0.9% NaCl. The goal of the dilutions is have the lipid concentrations high enough to give reliable optical density (OD) readings significantly above background, without exceeding or approaching the maximal OD reading of the plate reader. Thus, different batches of samples are diluted differently to meet these requirements.
d. For common inbred strains on a normal low fat chow diet, 75 µL of plasma is added to 225 µL of 0.9% saline for a 4 fold dilution, giving enough total diluted sample to run all of the assays in triplicate.
Preparing Standards for each assay
a. Standards for the various assays are initially prepared at the concentrations listed below for each assay.
b. Each standard is then diluted with 0.9% NaCl to the same fold dilution as the unknown plasma samples with the exception of the most concentrated cholesterol standard.
c. The most concentrated cholesterol standard (Standard 4 = 400 mg/dL) is diluted only half as much as the unknown plasma samples, and none of the FFA standards undergo further dilution after they are prepared.
Triglyceride (glycerol) standards: Standard 1 = 4.81 mg/dL glycerol (equivalent to 46.25 mg/dL TG), Standard 2 = 9.62 mg/dL glycerol (equivalent to 92.5mg/dL TG), Standard 3 = 19.24 mg/dL glycerol (equivalent to 185 mg/dL TG) and Standard 4 = 38.48 mg/dL glycerol (equivalent to 370 mg/dL TG).
Cholesterol Standards: Standards 5 and 4 are prepared by taking the cholesterol standard (200 mg/dL) directly as provided.
1. Standards 3 through 1 are then prepared by serial two fold dilutions from standard 4.
2. Concentrations of the standards are Standard 1= 25 mg/dL, Standard 2 = 50 mg/dL, Standard 3 = 100 mg/dL, Standard 4 = 200 mg/dL, and Standard 5 = 400 mg/dL.
3. Standards 4 through 1 are diluted with 0.9% NaCl to the same fold dilution as the unknown samples, while Standard 5 undergoes only half the dilution of the unknowns.
Nonesterified fatty acid (FFA) standards: The FFA concentration in the standard solution is 28.25 mg/dL.
1. Standard 1 is the stock solution diluted 1.5 x of the sample dilution and is labeled 18.83 mg/dL.
2. Standard 2 is the stock at the same dilution as the samples and is labeled 28.25 mg/dL.
3. Standard 3 is the stock diluted 0.5x and is labeled 56.5 mg/dL.
Glucose standards: Standards are made at the following concentrations: Standard 1 = 100 mg/dL, Standard 2 = 200 mg/dL, and Standard 3 = 400 mg/dL.
1. Once the three standards are prepared, they are further diluted with 0.9% NaCl to the same extent as the unknown samples.
Preparing Control Samples
a. Two control samples are run, a low value (Control 1) and a high value (Control 2), for each assay on each 96-well plate. Control 1 is SER-T-FY-1 Level 1 human control serum and Control 2 is SER-T-FY-2 Level 2 human control serum.
b. The exact concentration of each analyte varies slightly by lot# and the specific values for a lot are included on the lot specification sheet with each shipment.
c. The values for each analyte for each control are generally in the following ranges:
Glucose: Control 1 = 95 mg/dL, Control 2 = 300 mg/dL
Cholesterol: Control 1 = 95 mg/dL, Control 2 = 300 mg/dL
Free fatty acids: Control 1 = 9 mg/dL, Control 2 = 40 mg/dL
HDL cholesterol: Control 1 = 95 mg/dL, Control 2 = 300 mg/dL
Preparing Assay blanks
a. Sample "blanks" are prepared for each assay by adding 75 µL of saline rather than plasma, to make the initial dilutions from which aliquots are taken for all of the assays.
b. This "blank" is then run exactly the same way as the unknown plasma samples for all of the assays (except HDL), and the "blank" OD reading (which should be essentially zero) is subtracted from all other values.
c. In addition to the saline "blank" described above, the HDL assay also includes a heparin-MnCl2 blank, since there is a heparin-MnCl2 precipitation step in the HDL assay.
d. In the case of the heparin-MnCl2 blank, 30 µL of the "supernatant" is taken just as the samples containing plasma, since there is no precipitate with the addition of saline instead of plasma.
e. This "blank" from the precipitation is used to subtract from the HDL cholesterol values obtained for the unknown and control samples, while the saline "blank" is used for the standards on the HDL assay.
Setup for individual assays
a. Additional step for HDL assay only: prior to running the cholesterol assay the HDL is isolated from the other lipoproteins by precipitation.
b. The cholesterol assays for total cholesterol and unesterified cholesterol are done directly on aliquots of the diluted plasma samples.
c. ApoB-containing lipoproteins are precipitated from 100 µL of the diluted plasma in the 96 well U-bottom plates using heparin-MnCl2.
d. Plates are then centrifuged for 30 min at 40°C at 2500 rpm, in a Beckman TJ-6 (or comparable) centrifuge.
e. Supernatants (30 µL) are then taken for the cholesterol assay. Since the plasma sample is diluted further in the heparin-MnCl2 precipitation step, the HDL cholesterol values are multiplied by 1.2.
a. Total cholesterol assay is done on 20 µL of the diluted plasma sample, the unesterified cholesterol (UC) assay on 25 µL of the diluted plasma sample, and the HDL assay on 30 µL of the supernatant after heparin-MnCl2 precipitation.
b. The samples, controls and standards are added to 0.65 mL minitubes to which 600 µL of the cholesterol reagent is added.
c. The reagent with esterase is used for the total cholesterol and HDL assays, and the reagent without esterase is used for the UC assay.
d. The samples are then incubated at 37°C in a water bath.
e. After the incubation 170 µL aliquots in triplicates are loaded into the 96 well flat bottom plates.
f. The plates are read at 515 nm, subtracting the "blank" values from all readings.
g. Because the total cholesterol is higher than unesterified or HDL cholesterol, standards 100 mg/dL, 200 mg/dL, and 400 mg/dL are used. For the HDL assays, standards 50 mg/dL, 100 mg/dL, and 200 mg/dL are used. For the UC assay standards 25 mg/dL, 50 mg/dL, and 100 mg/dL are used.
Triglyceride and triglyceride blank assays
a. For the triglyceride and triglyceride blank assays 30 µL of the diluted samples, controls, and standards are aliquoted into 0.65 mL minitubes.
b. For the triglyceride blank assay glycerol standards 46.25 mg/dL, 92.5 mg/dL, and 185 mg/dL are used and for the triglyceride assay glycerol standards 92.5 mg/dL, 185 mg/dL, and 370 mg/dL are used.
c. Triglyceride assay reagent (600 µL) is added with lipase to each tube for the triglyceride assays, and 600 µL of the triglyceride reagent is added without lipase to each tube for the triglyceride blank assay.
d. Tubes are incubated for 10 min in a 37°C water bath.
e. After incubation 170 µL in triplicates are loaded into 96 well flat bottom plates and are read at 540 nm.
f. After running the triglyceride and triglyceride blank assays for each sample, the value of the triglyceride blank is subtracted from the triglyceride value to correct for endogenous levels of free glycerol in the plasma.
Free Fatty acid (FFA) assays
a. Thirty microliters (30 µL) of the diluted plasma samples, controls, and standards are aliquoted into 0.65 mL minitubes.
b. Reagent A (400 µL) is added and then incubated for 5 min in a 37°C water bath.
c. Reagent B (200 µL) is added and then incubated for 5 min in a 37°C water bath.
d. After incubation 170 µL in triplicates are loaded into 96 well flat bottom plates and are read at 550 nm.
e. "Blank" values are subtracted from the reading.
a. Mice are measured for total body fat mass by nuclear magnetic resonance (NMR) using the Bruker Minispec with software from Echo Medical Systems.
b. Mice are gently taken out of their cages and identification recorded.
c. Each mouse is guided into a restrainer, eliminating the need for anesthesia.
d. While within the restrainer, the mouse is placed into the minispec probe.
e. Body composition data are obtained according to the manufacturer's protocol.
f. Following data acquisition, the mouse is removed from the restrainer for cleaning and preparation for the next mouse to be measured.
For more details, see UCLA Systems Genetics Resources
Hybrid mouse diversity panel (HMDP): consists of a population of over 100 inbred mouse strains selected for usage in systematic genetic analyses of complex traits; 29 classic parental inbred and panels of recombinant inbred (RI) mice, including the BXD, CXB, BXA/AXB, and BXH panels.
- HDL cholesterol (plasma HDL)
- low density lipoprotein (plasma LDL) plus very low density lipoprotein (plasma VLDL)
- unesterified cholesterol (plasma CHOL)
- total cholesterol (plasma CHOL)
- free fatty acids (plasma FFA)
- triglycerides (plasma TG)
- percent of tissue mass that is fat, head included