View source for 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==
#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.