Triglyceride Assay from Cells and Tissues

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) (to make 50 mL of homogenization buffer - 2.5 mL of 1M Tris pH 8, 500 uL of 0.5 M EDTA, 0.273 g Mannitol, fill up the rest with water)
• 10M KOH (28.1g in 50 mL of water)
• 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. Weigh out 30-50mg tissue (record weights for normalization) on dry ice into round bottom eppendorf tube (2 mL). Add one stainless steel ball bearing.
2. Add 500ul Homogenization Buffer
3. Homogenize with Qiagen Tissue Lyser for 3 minutes @ 25Hz for Liver/WAT or for 5 minutes @ 30Hz for muscle
4. Add 12.5ul KOH
5. Mix by inverting then transfewr sample to a new 1.5 mL tube. Place dirty ball bearing in ethanol (located in the fume hood)
6. Add 800ul Chloroform/Methanol Mixture
7. Vortex vigorously then sit at room temperature for 5 minutes
8. Centrifuge for 10 minutes @ 13000G
9. Transfer 200 ul of the bottom layer into a new tube
10. Centrifuge again for 7-10 minutes @ 13000G
11. Transfer 150 ul of the bottom layer into the new tube (if there is enough of the bottom layer, transfer a total of 200 ul)
12. Let evaporate overnight at room temperature
13. Add(50ul) of Butanol Mixture and vortex. See Suggested Volumes for your specific tissue.
14. Measure triglyceride levels using SIgma Diagnostic Kit, using 5ul of sample.
    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 80uL of glycerol reagent and 20uL of triglyceride reagent. Make extra and combine in a Falcon tube.
    4. Aliquot 100ul into a well of a 96 well plate
    5. For standards, add 0-5 and .5ul of glycerol standard
    6. Add 5ul of resuspended lipid to each well to start (also make a 5ul blank of the butanol mixture) and mix.
    7. Pop any bubbles with tip before incubating.
    8. 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.
    9. Measure absorbance @ 540nm
    10. If any samples are A540<0.1 or above the 5ul standard the A540, then repeat with more or less lipid as required.