Triglyceride Assay from Cells and Tissues: Difference between revisions

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==Materials==

* '''Homogenization Buffer''' (50 mM Tris, 5 mM EDTA, 30 mM Mannitol, PI inhibitor)

* '''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

* 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 ~~337-B~~)

* Sigma Triglyceride Assay Kit (Cat TR0100)

==Protocol==

# Weigh out ~~200~~-~~500mg~~ tissue (record ~~weight~~ for normalization)

#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.

# ~~Homogenize with dounce homogenizer in 2 mL '''~~Homogenization Buffer~~'''.~~

#Add 500ul Homogenization Buffer

# ~~Remove 200 uL to a tube containing~~ 5 uL KOH

#Homogenize with Qiagen Tissue Lyser for 3 minutes @ 25Hz for Liver/WAT or for 5 minutes @ 30Hz for muscle

# Mix by inverting

#Add 12.5ul KOH

# Add ~~800 uL~~ '''Chloroform/Methanol Mixture'''

#Mix by inverting then transfewr sample to a new 1.5 mL tube. Place dirty ball bearing in ethanol (located in the fume hood)

# Vortex vigorously then sit at room temperature for 5 ~~min~~

#Add 800ul '''Chloroform/Methanol Mixture'''

# Centrifuge ~~10min at 13 000 RPM~~

#Vortex vigorously then sit at room temperature for 5 minutes

# ~~Take 180 uL~~ of the bottom layer into a ~~fresh~~ tube.

#Centrifuge for 10 minutes @ 13000G

# ~~Dry in fume hood overnight (or until completely dry)~~

#Transfer 200 ul of the bottom layer into a new tube

# ~~If absorbance~~ is ~~going to be measured by cuvette~~, ~~use non-bolded values. If you are using~~ a ~~plate reader use bolded values.~~

#Centrifuge again for 7-10 minutes @ 13000G

# Add ~~50uL~~ '''(~~500uL~~)''' of '''Butanol Mixture'''

#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)

# Measure triglyceride levels using ~~Sigma~~ Diagnostic Kit using ~~5 uL~~ of sample:

#Let evaporate overnight at room temperature

## Resuspend triglyceride and glycerol reagent with water if necessary.

#Add'''(50ul)''' of '''Butanol Mixture''' and vortex. See Suggested Volumes for your specific tissue.

## Calculate how many ~~samples~~ you have (samples + ~~buffer~~ blank + 6 standard curve ~~values~~).

#Measure triglyceride levels using SIgma Diagnostic Kit, using 5ul of sample.

## 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.

##Resuspend triglyceride and glycerol reagent with water if necessary

## Aliquot ~~700 uL into a cuvette or~~ '''~~100 uL~~ into a well of a 96 well plate'''.

##Calculate how many sample you have (samples + blank + standard curve)

## For standards add 0-5 uL of glycerol standard ~~'''(or of a 1/10 dilution of the glycerol standard)'''.~~

##Prepare reagent. You need 80uL of glycerol reagent and 20uL of triglyceride reagent. Make extra and combine in a Falcon tube.

## Add ~~5 uL~~ of resuspended lipid to each well to start (also make a ~~5uL~~ blank of the butanol mixture) and mix.

##Aliquot '''100ul into a well of a 96 well plate'''

## Let sit for ~30 ~~min at~~ room temperature (or ~~5 min at~~ 37C if you are in a hurry).

##For standards, add 0-5 and .5ul of glycerol standard

## Measure absorbance ~~at 540 nm.~~

##Add 5ul of resuspended lipid to each well to start (also make a 5ul blank of the butanol mixture) and mix.

## If any samples are A540<0.1 or above the ~~5uL~~ standard A540 then repeat with more or less lipid as required.

##Pop any bubbles with tip before incubating.

##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.

@@ Line 1: / Line 1: @@
 ==Materials==
-* '''Homogenization Buffer''' (50 mM Tris, 5 mM EDTA, 30 mM Mannitol, PI inhibitor)
+* '''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
+* 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 337-B)
+* Sigma Triglyceride Assay Kit (Cat TR0100)
 ==Protocol==
-# Weigh out 200-500mg tissue (record weight for normalization)
+#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.
-# Homogenize with dounce homogenizer in 2 mL '''Homogenization Buffer'''.
+#Add 500ul Homogenization Buffer
-# Remove 200 uL to a tube containing 5 uL KOH
+#Homogenize with Qiagen Tissue Lyser for 3 minutes @ 25Hz for Liver/WAT or for 5 minutes @ 30Hz for muscle
-# Mix by inverting
+#Add 12.5ul KOH
-# Add 800 uL '''Chloroform/Methanol Mixture'''
+#Mix by inverting then transfewr sample to a new 1.5 mL tube. Place dirty ball bearing in ethanol (located in the fume hood)
-# Vortex vigorously then sit at room temperature for 5 min
+#Add 800ul '''Chloroform/Methanol Mixture'''
-# Centrifuge 10min at 13 000 RPM
+#Vortex vigorously then sit at room temperature for 5 minutes
-# Take 180 uL of the bottom layer into a fresh tube.
+#Centrifuge for 10 minutes @ 13000G
-# Dry in fume hood overnight (or until completely dry)
+#Transfer 200 ul of the bottom layer into a new tube
-# If absorbance is going to be measured by cuvette, use non-bolded values.  If you are using a plate reader use bolded values.
+#Centrifuge again for 7-10 minutes @ 13000G
-# Add 50uL '''(500uL)''' of '''Butanol Mixture'''
+#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)
-# Measure triglyceride levels using Sigma Diagnostic Kit using 5 uL of sample:
+#Let evaporate overnight at room temperature
-## Resuspend triglyceride and glycerol reagent with water if necessary.
+#Add'''(50ul)''' of '''Butanol Mixture''' and vortex. See Suggested Volumes for your specific tissue.
-## Calculate how many samples you have (samples + buffer blank + 6 standard curve values).
+#Measure triglyceride levels using SIgma Diagnostic Kit, using 5ul of sample.
-## 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.
+##Resuspend triglyceride and glycerol reagent with water if necessary
-## Aliquot 700 uL into a cuvette or '''100 uL into a well of a 96 well plate'''.
+##Calculate how many sample you have (samples + blank  + standard curve)
-## For standards add 0-5 uL of glycerol standard '''(or of a 1/10 dilution of the glycerol standard)'''.
+##Prepare reagent. You need 80uL of glycerol reagent and 20uL of triglyceride reagent. Make extra and combine in a Falcon tube.
-## Add 5 uL of resuspended lipid to each well to start (also make a 5uL blank of the butanol mixture) and mix.
+##Aliquot '''100ul into a well of a 96 well plate'''
-## Let sit for ~30 min at room temperature (or 5 min at 37C if you are in a hurry).
+##For standards, add 0-5 and .5ul of glycerol standard
-## Measure absorbance at 540 nm.
+##Add 5ul of resuspended lipid to each well to start (also make a 5ul blank of the butanol mixture) and mix.
-## If any samples are A540<0.1 or above the 5uL standard A540 then repeat with more or less lipid as required.
+##Pop any bubbles with tip before incubating.
+##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.