Liver Cholesterol and Triglycerides Measurements

Liver Cholesterol and Triglycerides Measurements 

Reagents:

Matrix Plus™ Chemistry Reference Kit (Triglycerides standard), Verichem Laboratories, Cat#: 9500

Matrix Plus™ Cholesterol Reference Kit, Verichem Laboratories, Cat#: 9550

Thermo Scientific™ Total Cholesterol Reagents. Fisher Scientific, Cat#: TR13421

Thermo Scientific™ Triglycerides Reagents. Fisher Scientific, Cat#: TR22421

Procedures:

1. Weigh 80 – 100 mg of liver and place into a 2-mL tube round bottom tube, record the weight of the liver sample used. 
2. Add 1ml of 2:1 chloroform:methanol mix (Folch) to the tube.
3. Homogenize the liver sample with Tissuelyser.
4. Spin the sample at 10,000 g for 5 minutes at room temperature with a tabletop centrifuge.
5. Transfer the supernatant from step 4 to a 16x125 mm disposable glass tube.
6. Add 1ml of Folch to the pellet, repeat step 3-5, and transfer the supernatant to the same tube in step 5.
7. Add 1 mL of PBS to the tube with supernatant, and vortex vigorously.
8. Centrifuge at 1,000 g for 10 min at room temperature.
9. Transfer the bottom layer to a new 16x100 mm glass tube with cap.
10. Add 2 mL of Chloroform to the 16x125 mm tube with remaining top layer sample and vortex vigorously.
11. Centrifuge at 1,000 g for 10 min at room temperature and transfer the bottom layer to the same 16x100 mm in step 9.
12. Transfer the bottom layer to the 16x100 mm glass tubes with caps and adjust lipid extract to 5 mL with chloroform.
13. In 12x75 mm glass tubes, add 10µl of Trition in chloroform (2:1) using an organic pipette to the bottom of each tube for standards and samples. 
14. Add standards and samples to the glass tubes
15. Total Cholesterol: 
    1. 4 µl of standards 
    2. 200 µl of lipid extract from step 12
16. Triglyceride: 
    1. 4 µl of standards 
    2. 50 µl of lipid extract from step 12
17. Vortex each tube vigorously.
18. Dry out the tubes under regular air at 37^oC
19. Add 300µl of TG or Cholesterol reagent.
20. Vortex the tubes vigorously for 1 -2 min and incubate in 37°C for 5 min
21. Read the absorption with a plate reader according to the reagent manual.