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
These volumes are for a well of a 6 well plate.
Aspirate media
Wash cells with 1 mL of ice cold PBS after treatment (aspirate).
Add 200ul Homogenization Buffer and transfer to microtube
Homogenize by sonication (3 x 15s) or 3 x freeze/thaw cycles in liquid nitrogen
Add 5ul KOH
Mix by inverting
Add 800ul Chloroform/Methanol Mixture
Vortex vigorously then sit at room temperature for 5 minutes
Centrifuge at 4 degrees for 10 minutes @ 13000G
Transfer the bottom layer into a new tube
Let evaporate overnight at room temperature
If absorbance is going to be measured by cuvette, use non-bolded values. If using a plate reader, used bolded values.
Add 500ul (50ul) of Butanol Mixture. See Suggested Volumes for your specific tissue.
Measure triglyceride levels using SIgma Diagnostic Kit, using 5ul of sample/butanol mix.
Resuspend triglyceride and glycerol reagent with water if necessary
Calculate how many sample you have (samples + blank + standard curve)
Prepare reagent. You need 560ul (80ul) of glycerol reagent and 140ul (20ul) of triglyceride reagent. Make extra and combine in a Falcon tube.
Aliquot 700ul into a cuvette or 100ul into a well of a 96 well plate
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).
Add 5ul of resuspended lipid to each well to start (also make a 5ul blank of the butanol mixture) and mix.
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.
Measure absorbance @ 540nm
If any samples are A540<0.1 or above the 5ul standard the A540, then repeat with more or less lipid as required.