Difference between revisions of "Triglyceride Assay from Tissue Culture 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. | ||
Revision as of 18:11, 5 August 2014
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 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.
