|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.
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.]
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.
Sample preparation for HDL cholesterol (Modified Beckman
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
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
2. Run samples.
3. Remove and discard cups once all tests have been run.