Triglyceride Assay from Cells and Tissues

Materials

• Homogenization Buffer (50 mM Tris, 5 mM EDTA, 30 mM Mannitol, PI inhibitor)
• 10M KOH
• Chloroform/Methanol Mixture (2:1)
• Butanol Mixture: 3 mL butanol, 1.66 mL Triton-X114, 0.33 mL Methanol
• Sigma Triglyceride Assay Kit (Cat 337-B)

Protocol

1. Weigh out 200-500mg tissue (record weight for normalization)
2. Homogenize with dounce homogenizer in 2 mL Homogenization Buffer.
3. Remove 200 uL to a tube containing 5 uL KOH
4. Mix by inverting
5. Add 800 uL Chloroform/Methanol Mixture
6. Vortex vigorously then sit at room temperature for 5 min
7. Centrifuge 10min at 13 000 RPM
8. Take 180 uL of the bottom layer into a fresh tube.
9. Dry in fume hood overnight (or until completely dry)
10. If absorbance is going to be measured by cuvette, use non-bolded values. If you are using a plate reader use bolded values.
11. Add 50uL (500uL) of Butanol Mixture
12. Measure triglyceride levels using Sigma Diagnostic Kit using 5 uL of sample:
    1. Resuspend triglyceride and glycerol reagent with water if necessary.
    2. Calculate how many samples you have (samples + buffer blank + 6 standard curve values).
    3. Prepare reagent, you need 560 uL (80uL) of glycerol reagent and 140 uL (20uL) of triglyceride reagent. Make a bit extra and combine in a falcon tube.
    4. Aliquot 700 uL into a cuvette or 100 uL into a well of a 96 well plate.
    5. For standards add 0-5 uL of glycerol standard (or of a 1/10 dilution of the glycerol standard).
    6. Add 5 uL of resuspended lipid to each well to start (also make a 5uL blank of the butanol mixture) and mix.
    7. Let sit for ~30 min at room temperature (or 5 min at 37C if you are in a hurry).
    8. Measure absorbance at 540 nm.
    9. If any samples are A540<0.1 or above the 5uL standard A540 then repeat with more or less lipid as required.