Paigen3 project protocol

Diet effects on gallstone formation, liver pathology, plasma lipids, and pathogen-accelerated atherosclerosis in 8 obesity mutant mouse strains   (2000)

Paigen B, Bouchard G, Carey MC
With: Johnson D, Tovbina M, Silva A, Carver T, Tracy T, McFarland C, Archer J, Whitmore H




This project is part of the Heart, Lung, Blood and Sleep Disorders Consortium. This page describes protocols used for total cholesterol, HDL cholesterol, triglyceride, and glucose measurements. Naggert1 contains glucose data for mice on a a high fat, high cholesterol atherogenic diet. Paigen2 contains lipid measurements taken before and after ingesting the high-fat diet. Additional baseline lipid data on 10 wk old mice is found in Paigen4. Procedures for assessing aortic lesions can be found in the Paigen1 protocol.


Cholesterol, triglyceride, glucose



Conditions: Mice were shipped from JAX production facility at 6-7 wks of age and fed LabDiet 5K52 (6% fat) during the acclimation period. Samples were collected for baseline testing from mice at 7-10 wks of age. A set of these mice were then administered a high fat, high cholesterol atherogenic diet for 17 weeks and retested. Prior to blood collection, mice were put under fasting conditions for 4 hours, beginning one hour after the start of the light cycle (food withheld at 0700 h; blood drawn at 1100 h).

Sample collection: Approximately 225 uL of blood (6-8 drops plus one full capillary tube) were collected from each mouse (depending on its size) via a retro-orbital bleed using heparin-coated hematocrit tubes. The blood was collected into 1.5 mL microfuge tubes containing 7.5 uL of Heparin 1000 U/mL (Sigma-Aldrich #H3393) and put on ice immediately. Samples were centrifuged at 14,000 rpm for 5 min. A minimum of 100 uL of blood plasma was collected from the upper layer, leaving the blood cells behind in the microfuge tube, which was discarded. The plasma was transferred into a fresh 0.5 mL microfuge tube and frozen for later analysis.

Equipment: Beckman Coulter Synchron CX5 (Chemistry Analyzer). A dedicated DOS-based desktop computer controls the programming of the Chemistry Analyzer. A dedicated printer prints the results as they are measured, and an electronic file is simultaneously transferred to a second, Windows-based computer, which stores the data files.

Reagents and Expendables: The following supplies and quantities were used for each sample tested (for all four of the above chemistries): 1.5 uL microfuge tube (1), 0.5 mL Beckman Coulter MicrotubeTM Tubecup ("sector cup") (2), and 200 uL pipette tips (3-4, depending on whether dilution is necessary).

The Chemistry Analyzer uses Beckman Coulter three-compartment reagent cartridges for HDLC, CHOL, TG, and GLU.

ReagentBeckman Coulter Reorder Number
CHOL 467825
GLU 442640
TG 445850

Each cartridge contains enough reagent for 300 tests (approx. 104 mL). In addition, in order to run the HDLC test, HDL Cholesterol Separation Reagent (15 uL per sample) is needed. The bottle from Beckman Coulter contains a volume of 34 mL. [If the dilution of plasma samples becomes necessary due to low plasma volume, 0.9% saline solution is used for the dilution.]

Calibration reagents
Calibrated commercial controls were run each day of testing (must be within established ranges prior to analyzing samples). The two calibration reagents are "Synchron® Systems HDL Cholesterol Calibrator" (for HDLC only), and "Synchron® Systems Multi Calibrator" (for CHOL, GLU, and TG).

Controls
The controls for HDLC are Beckman CoulterTM VigilTM Lipid Control 1 and Beckman CoulterTM VigilTM Lipid Control 2. The controls for CHOL, GLU and TG are Synchron® Control Comprehensive Chemistry Control Serum Level 1, Level 2, and Level 3.


Order of testing:

    1. HDL cholesterol (HDLC)

    2. total cholesterol (CHOL) & glucose (GLU)

    3. triglyceride (TG)

The HDLC test must be run separately because it utilizes a reagent to separate the two types of cholesterol from one another. The CHOL, GLU, and TG tests can be run at the same time. The plasma used for the HDLC test ends up with a dilution factor of 2 during processing (see below). If there is not enough plasma to run the other three tests, the plasma may be diluted with 0.9% saline.


Preparation:

Reagent preparation
Reagent cartridges are changed and calibrated as necessary.

Control preparation
Controls are run using the same procedure used for samples described below. The Lipid Controls (Levels 1 and 2) used for HDLC must be reacted with the HDL Cholesterol Separation Reagent, spun down, and extracted in the same manner, whereas the Comprehensive Chemistry Control Serum Levels 1, 2 and 3 are used directly.

Sample preparation for HDL cholesterol (Modified Beckman protocol).
This procedure uses six sectors (42 samples). If more or fewer samples need to be run, modify the procedure appropriately.

    1. Arrange 42 empty unmarked 1.5 mL microfuge tubes in a 96-place rack. Orient the rack as 12 rows x 8 columns, and skip every other row, placing seven tubes per row.

    2. Record the plasma identifying information (mouse I.D. number), and assign a Beckman Coulter sector and cell number to each sample, making sure that the sector numbers assigned are actually available for imminent use.

    3. Using a 250 uL repeating pipettor, add 35 uL of polyethylene glycol (20% PEG 8000 in 0.2 M glycine) to each open tube.

    4. Dispense 35 uL of plasma into the bottom of each tube (dilutes plasma; dilution factor=2). Change tips between each plasma sample. Close the caps.

    5. Label the caps with sector and cell number.

    6. Vortex each microfuge tube for 10-15 s to mix solutions.

    7. Let tubes sit for 15 min at room temperature.

    8. Place the tubes sequentially into a refrigerated microcentrifuge set at 4°C with the hinges pointing out. Centrifuge for 10 min at 14,000 rpm.

    9. Carefully remove the tubes from the centrifuge and replace them in the rack in the same six rows from which they were removed.

    10. Carefully draw off the supernatant, avoiding contact with the pellet, and transfer to fresh 0.5 mL Beckman Coulter MicrotubeTM Tubecups in the corresponding sector cup. Discard the tubes containing the pellets.

Sample preparation for total cholesterol, triglycerides, and glucose:
(these tests do not require the PEG step)

    1. Record the plasma identifying information (mouse I.D. number) and assign a Beckman Coulter sector and cell number to each sample, making sure that the sector numbers assigned are actually available for imminent use.

    2. Place seven new empty 0.5 mL Beckman Coulter MicrotubeTM Tubecups (sector cups) into each sector, one in each cup holder.

    3. Add 35 uL of 0.9% saline into the bottom of each Tubecup. Then add 35 uL of plasma into each assigned cup, giving a plasma dilution factor of 2. Minimum sample volume is 54 uL and 60-65 uL is preferred. Dilute samples further if necessary.

    4. Cap and number the tubes according to sector and cups.




Loading and running samples
    1. Program the Chemistry Analyzer using manufacturer's procedure.

    2. Run samples.

    3. Remove and discard cups once all tests have been run.




Investigator notes:
  • Be sure to plan the sequence and timing of all tasks carefully. There are numerous procedures within this protocol that may not be stopped in the middle, such as the HDLC sample preparation, followed immediately by the HDLC test. The reagent cartridges in the Chemistry Analyzer need to be changed regularly which interrupts the run. The cartridge needs to be changed, calibrations done, controls run, and then the run of samples can be resumed.
  • It is important to avoid introducing air bubbles into the sample, as these will interfere with the results or cause the Chemistry Analyzer to be unable to detect the sample. To minimize introduction of air bubbles into the sample, carefully position the pipette tip at the bottom of the sector cup and raise the tip up the side of the sector cup as the plunger is being depressed, taking care not to inject air into the sample once all the liquid has been expelled. Often one air bubble will remain in spite of these precautions - this can be popped using the wooden sticks that come with the Chemistry Analyzer or a toothpick.
  • Occasionally, the precipitate will not form a pellet at the bottom of the tube, or the pellet will break apart easily. In these cases, there will be opaque particles floating on or suspended in the plasma sample. The presence of these particles will often cause the HDLC result to be suppressed by the Beckman Coulter Chemistry Analyzer. To avoid this problem, try to position the pipette tip in such a way as to draw up only clear plasma, leaving the particles behind in the microfuge tube. Even when this procedure is attempted, it can still be quite difficult in certain cases to successfully separate the plasma from the suspended particles.
  • The cups in each sector are numbered from right to left. Be sure to load the samples into the correctly numbered sector cups.