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→Protocol
==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 Bufferand transfer to microtube#Homogenize by sonication (3 x 15s) or 3 x freeze/thaw cyclesin 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 glycerols 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.