Triglyceride Assay from Cells and Tissues: Difference between revisions

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==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)

* '''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

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==Protocol==

#~~Use 5 female or 8 Male flies~~ for ~~Assay~~

#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 ~~flies (~~3 ~~min~~ @ ~~40Hz)~~ in ~~1mL of .05% Tween~~ (~~from Tween-20 stock~~)

#Add 500ul Homogenization Buffer

#~~Heat samples in 70C waterbath~~ for 5 minutes

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

#~~Spin samples~~ @ ~~5000G for 1 minute~~

#Add 12.5ul KOH

#Transfer ~~500ul~~ of ~~supernatent in~~ new ~~microfuge~~ tube

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

#~~Spin @ 14000G~~ for 3 minutes

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

#Add 50ul of sample ~~to 200ul~~ of ~~TG solution~~ in a 96 well plate

#Vortex vigorously then sit at room temperature for 5 minutes

#~~Incubate plate~~ @ 37C ~~for 5 mins~~

#Centrifuge for 10 minutes @ 13000G

#Measure 540nm ~~absorbance~~

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

#Centrifuge again for 7-10 minutes @ 13000G

#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)

#Let evaporate overnight at room temperature

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

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

##Resuspend triglyceride and glycerol reagent with water if necessary

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

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

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

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

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

##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 pH 8, 5 mM EDTA, 30 mM Mannitol, PI inhibitor, can be made in bulk without the PI, PI added fresh)
+* '''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
@@ Line 7: / Line 7: @@
 ==Protocol==
-#Use 5 female or 8 Male flies for Assay
+#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 flies (3 min @ 40Hz) in 1mL of .05% Tween (from Tween-20 stock)
+#Add 500ul Homogenization Buffer
-#Heat samples in 70C waterbath for 5 minutes
+#Homogenize with Qiagen Tissue Lyser for 3 minutes @ 25Hz for Liver/WAT or for 5 minutes @ 30Hz for muscle
-#Spin samples @ 5000G for 1 minute
+#Add 12.5ul KOH
-#Transfer 500ul of supernatent in new microfuge tube
+#Mix by inverting then transfewr sample to a new 1.5 mL tube. Place dirty ball bearing in ethanol (located in the fume hood)
-#Spin @ 14000G for 3 minutes
+#Add 800ul '''Chloroform/Methanol Mixture'''
-#Add 50ul of sample to 200ul of TG solution in a 96 well plate
+#Vortex vigorously then sit at room temperature for 5 minutes
-#Incubate plate @ 37C for 5 mins
+#Centrifuge for 10 minutes @ 13000G
-#Measure 540nm absorbance
+#Transfer 200 ul of the bottom layer into a new tube
+#Centrifuge again for 7-10 minutes @ 13000G
+#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)
+#Let evaporate overnight at room temperature
+#Add'''(50ul)''' of '''Butanol Mixture''' and vortex. See Suggested Volumes for your specific tissue.
+#Measure triglyceride levels using SIgma Diagnostic Kit, using 5ul of sample.
+##Resuspend triglyceride and glycerol reagent with water if necessary
+##Calculate how many sample you have (samples + blank  + standard curve)
+##Prepare reagent. You need 80uL of glycerol reagent and 20uL of triglyceride reagent. Make extra and combine in a Falcon tube.
+##Aliquot '''100ul into a well of a 96 well plate'''
+##For standards, add 0-5 and .5ul of glycerol standard
+##Add 5ul of resuspended lipid to each well to start (also make a 5ul blank of the butanol mixture) and mix.
+##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.