Triglyceride Assay from Tissue Culture Cells: Difference between revisions
wrote protocol for cells |
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==Protocol== | ==Protocol== | ||
#These volumes are for a well of a 6 well plate. | #These volumes are for a well of a 6 well plate. | ||
#Wash cells with 1 mL of ice cold PBS after treatment | #Aspirate media | ||
#Add 200ul Homogenization Buffer | #Wash cells with 1 mL of ice cold PBS after treatment (aspirate). | ||
#Homogenize by sonication (3 x 15s) or 3 x freeze/thaw cycles | #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 | #Add 5ul KOH | ||
#Mix by inverting | #Mix by inverting | ||
#Add 800ul '''Chloroform/Methanol Mixture''' | #Add 800ul '''Chloroform/Methanol Mixture''' | ||
#Vortex vigorously then sit at room temperature for 5 minutes | #Vortex vigorously then sit at room temperature for 5 minutes | ||
#Centrifuge for 10 minutes @ 13000G | #Centrifuge at 4 degrees for 10 minutes @ 13000G | ||
#Transfer the bottom layer into a new tube | #Transfer the bottom layer into a new tube | ||
#Let evaporate overnight at room temperature | #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. | #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. | #Add 500ul '''(50ul)''' of '''Butanol Mixture'''. See Suggested Volumes for your specific tissue. | ||
#Measure triglyceride levels using SIgma Diagnostic Kit, using 5ul of sample. | #Measure triglyceride levels using SIgma Diagnostic Kit, using 5ul of sample/butanol mix. | ||
##Resuspend triglyceride and glycerol reagent with water if necessary | ##Resuspend triglyceride and glycerol reagent with water if necessary | ||
##Calculate how many sample you have (samples + blank + standard curve) | ##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. | ##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''' | ##Aliquot 700ul into a cuvette or '''100ul into a well of a 96 well plate''' | ||
##For standards, add 0-5ul of glycerol standard | ##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. | ##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. | ##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 | ##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. | ##If any samples are A540<0.1 or above the 5ul standard the A540, then repeat with more or less lipid as required. | ||