Triglyceride Assay from Tissue Culture Cells

Materials

• Homogenization Buffer (50 mM Tris pH 8, 5 mM EDTA, 30 mM Mannitol, PI inhibitor, can be made in bulk without the PI, PI added fresh)
• 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 TR0100)

Protocol

1. These volumes are for a well of a 6 well plate.
2. Aspirate media
3. Wash cells with 1 mL of ice cold PBS after treatment (aspirate).
4. Add 200ul Homogenization Buffer and transfer to microtube
5. Homogenize by sonication (3 x 15s) or 3 x freeze/thaw cycles in liquid nitrogen
6. Add 5ul KOH
7. Mix by inverting
8. Add 800ul Chloroform/Methanol Mixture
9. Vortex vigorously then sit at room temperature for 5 minutes
10. Centrifuge at 4 degrees for 10 minutes @ 13000G
11. Transfer the bottom layer into a new tube
12. Let evaporate overnight at room temperature
13. If absorbance is going to be measured by cuvette, use non-bolded values. If using a plate reader, used bolded values.
14. Add 500ul (50ul) of Butanol Mixture. See Suggested Volumes for your specific tissue.
15. Measure triglyceride levels using SIgma Diagnostic Kit, using 5ul of sample/butanol mix.
    1. Resuspend triglyceride and glycerol reagent with water if necessary
    2. Calculate how many sample you have (samples + blank + standard curve)
    3. Prepare reagent. You need 560ul (80ul) of glycerol reagent and 140ul (20ul) of triglyceride reagent. Make extra and combine in a Falcon tube.
    4. Aliquot 700ul into a cuvette or 100ul into a well of a 96 well plate
    5. For standards, add 0-5ul of glycerol standard (standard 1-1ul of glycerol standard, 2-2ul, etc.)---If you have reason to believe your signal is low, or if you do not have an idea of the level of signal of your samples you may want to include a second set of diluted standards. In this case you should make a 1:10 dilution of the glycerol standard and water and add 4, 6, 8, and 10ul of that).
    6. Add 5ul of resuspended lipid to each well to start (also make a 5ul blank of the butanol mixture) and mix.
    7. Let sit for ~30 mins @ room temperature (or 5mins @ 37C if you are in a hurry). If using >10ul of butanol mix the solution may be cloudy. Let it settle and it should become more clear.
    8. Measure absorbance @ 540nm
    9. If any samples are A540<0.1 or above the 5ul standard the A540, then repeat with more or less lipid as required.