Difference between revisions of "Triglyceride Assay from Cells and Tissues"

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==Materials==
 
==Materials==
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* '''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)
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* '''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) (to make 50 mL of homogenization buffer - 2.5 mL of 1M Tris pH 8, 500 uL of 0.5 M EDTA, 0.273 g Mannitol, fill up the rest with water)
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* 10M KOH
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* 10M KOH (28.1g in 50 mL of water)
 
* '''Chloroform/Methanol Mixture''' (2:1)
 
* '''Chloroform/Methanol Mixture''' (2:1)
 
* '''Butanol Mixture''': 3 mL butanol, 1.66 mL Triton-X114, 0.33 mL Methanol
 
* '''Butanol Mixture''': 3 mL butanol, 1.66 mL Triton-X114, 0.33 mL Methanol
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==Protocol==
 
==Protocol==
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# Weigh out 200-500mg tissue (record weight for normalization).  You can use as low as 30 mg tissue if necessary by reducing the lysis volume (must be at least 500 uL) or increasing the amount of the lower (chloroform) layer removed up to 500 ul(normally 180 uL).
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#Weigh out 30-50mg tissue (record weights for normalization) on dry ice into round bottom eppendorf tube (2 mL). Add one stainless steel ball bearing.
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# Homogenize with dounce homogenizer in 2 mL '''Homogenization Buffer'''.
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#Add 500ul Homogenization Buffer
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# Remove 200 uL to a tube containing 5 uL KOH
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#Homogenize with Qiagen Tissue Lyser for 3 minutes @ 25Hz for Liver/WAT or for 5 minutes @ 30Hz for muscle
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# Mix by inverting
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#Add 12.5ul KOH
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# Add 800 uL '''Chloroform/Methanol Mixture'''
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#Mix by inverting then transfewr sample to a new 1.5 mL tube. Place dirty ball bearing in ethanol (located in the fume hood)
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# Vortex vigorously then sit at room temperature for 5 min
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#Add 800ul '''Chloroform/Methanol Mixture'''
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# Centrifuge 10min at 13 000 RPM
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#Vortex vigorously then sit at room temperature for 5 minutes
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# Take 200 uL of the bottom layer into a fresh tube.  See [[#Suggested Volumes | Suggested Volumes]] for your specific tissue
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#Centrifuge for 10 minutes @ 13000G
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# Dry in fume hood overnight (or until completely dry)
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#Transfer 200 ul of the bottom layer into a new tube
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# If absorbance is going to be measured by cuvette, use non-bolded values.  If you are using a plate reader use bolded values.
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#Centrifuge again for 7-10 minutes @ 13000G
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# Add 50uL '''(500uL)''' of '''Butanol Mixture'''. See [[#Suggested Volumes | Suggested Volumes]] for your specific tissue
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#Transfer 150 ul of the bottom layer into the new tube (if there is enough of the bottom layer, transfer a total of 200 ul)
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# Measure triglyceride levels using Sigma Diagnostic Kit using 5 uL of sample:
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#Let evaporate overnight at room temperature
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## Resuspend triglyceride and glycerol reagent with water if necessary.
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#Add'''(50ul)''' of '''Butanol Mixture''' and vortex. See Suggested Volumes for your specific tissue.
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## Calculate how many samples you have (samples + buffer blank + 6 standard curve values).
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#Measure triglyceride levels using SIgma Diagnostic Kit, using 5ul of sample.
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## Prepare reagent, you need 560 uL '''(80uL)''' of glycerol reagent and 140 uL '''(20uL)''' of triglyceride reagent. Make a bit extra and combine in a falcon tube.
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##Resuspend triglyceride and glycerol reagent with water if necessary
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## Aliquot 700 uL into a cuvette or '''100 uL into a well of a 96 well plate'''.
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##Calculate how many sample you have (samples + blank + standard curve)
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## For standards add 0-5 uL of glycerol standard '''(or of a 1/10 dilution of the glycerol standard)'''.
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##Prepare reagent. You need 80uL of glycerol reagent and 20uL of triglyceride reagent. Make extra and combine in a Falcon tube.
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## Add 5 uL of resuspended lipid to each well to start (also make a 5uL blank of the butanol mixture) and mix.
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##Aliquot '''100ul into a well of a 96 well plate'''
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## Let sit for ~30 min at room temperature (or 5 min at 37C if you are in a hurry). If using > 10 uL of butanol mixture the solution may be cloudy, let it settle and it should become more clear.
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##For standards, add 0-5 and .5ul of glycerol standard
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## Measure absorbance at 540 nm.
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##Add 5ul of resuspended lipid to each well to start (also make a 5ul blank of the butanol mixture) and mix.
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## If any samples are A540<0.1 or above the 5uL standard A540 then repeat with more or less lipid as required.
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##Pop any bubbles with tip before incubating.
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##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.
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===Suggested Volumes===
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##Measure absorbance @ 540nm
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This is based on using a 96 well plate to measure final concentrations.
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##If any samples are A540<0.1 or above the 5ul standard the A540, then repeat with more or less lipid as required.
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{| border="1"
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−
|-
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! Tissue/Condition !! Lysis Volume !! Chloroform Volume !! Resuspension Volume !! Assay Volume
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−
|-
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| Liver || 1 mL || 200 uL || 500 uL || 5 uL
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−
|-
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| Muscle || 1 mL || 200 uL || 50 uL || 5 uL
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|}
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Latest revision as of 17:03, 11 October 2019

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) (to make 50 mL of homogenization buffer - 2.5 mL of 1M Tris pH 8, 500 uL of 0.5 M EDTA, 0.273 g Mannitol, fill up the rest with water)
  • 10M KOH (28.1g in 50 mL of water)
  • 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

  1. Weigh out 30-50mg tissue (record weights for normalization) on dry ice into round bottom eppendorf tube (2 mL). Add one stainless steel ball bearing.
  2. Add 500ul Homogenization Buffer
  3. Homogenize with Qiagen Tissue Lyser for 3 minutes @ 25Hz for Liver/WAT or for 5 minutes @ 30Hz for muscle
  4. Add 12.5ul KOH
  5. Mix by inverting then transfewr sample to a new 1.5 mL tube. Place dirty ball bearing in ethanol (located in the fume hood)
  6. Add 800ul Chloroform/Methanol Mixture
  7. Vortex vigorously then sit at room temperature for 5 minutes
  8. Centrifuge for 10 minutes @ 13000G
  9. Transfer 200 ul of the bottom layer into a new tube
  10. Centrifuge again for 7-10 minutes @ 13000G
  11. Transfer 150 ul of the bottom layer into the new tube (if there is enough of the bottom layer, transfer a total of 200 ul)
  12. Let evaporate overnight at room temperature
  13. Add(50ul) of Butanol Mixture and vortex. See Suggested Volumes for your specific tissue.
  14. Measure triglyceride levels using SIgma Diagnostic Kit, using 5ul of sample.
    1. Resuspend triglyceride and glycerol reagent with water if necessary
    2. Calculate how many sample you have (samples + blank + standard curve)
    3. Prepare reagent. You need 80uL of glycerol reagent and 20uL of triglyceride reagent. Make extra and combine in a Falcon tube.
    4. Aliquot 100ul into a well of a 96 well plate
    5. For standards, add 0-5 and .5ul of glycerol standard
    6. Add 5ul of resuspended lipid to each well to start (also make a 5ul blank of the butanol mixture) and mix.
    7. Pop any bubbles with tip before incubating.
    8. 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.
    9. Measure absorbance @ 540nm
    10. If any samples are A540<0.1 or above the 5ul standard the A540, then repeat with more or less lipid as required.
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