Triglyceride Assay from Cells and Tissues - Revision history

Kkistler at 17:03, 11 October 2019

2019-10-11T17:03:31Z

← Older revision		Revision as of 17:03, 11 October 2019
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	#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 for 10 minutes @ 13000G
	#Transfer ~~400~~ ul of the bottom layer into a new tube		#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		#Let evaporate overnight at room temperature
	#Add'''(50ul)''' of '''Butanol Mixture''' and vortex. See Suggested Volumes for your specific tissue.		#Add'''(50ul)''' of '''Butanol Mixture''' and vortex. See Suggested Volumes for your specific tissue.

Kkistler at 18:23, 1 October 2019

2019-10-01T18:23:23Z

← Older revision		Revision as of 18:23, 1 October 2019
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	==Materials==		==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)		* '''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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	##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 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'''		##Aliquot '''100ul into a well of a 96 well plate'''
	##For standards, add 0-5 and .5ul of glycerol standard		##For standards, add 0-5 and .5ul of glycerol standard

Pfeiferl at 17:05, 7 June 2017

2017-06-07T17:05:21Z

← Older revision		Revision as of 17:05, 7 June 2017
Line 17:		Line 17:
	#Transfer 400 ul of the bottom layer into a new tube		#Transfer 400 ul of the bottom layer into a new tube
	#Let evaporate overnight at room temperature		#Let evaporate overnight at room temperature
	#Add'''(50ul)''' of '''Butanol Mixture'''. See Suggested Volumes for your specific tissue.		#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.		#Measure triglyceride levels using SIgma Diagnostic Kit, using 5ul of sample.
	##Resuspend triglyceride and glycerol reagent with water if necessary		##Resuspend triglyceride and glycerol reagent with water if necessary

Pfeiferl at 17:04, 7 June 2017

2017-06-07T17:04:46Z

← Older revision		Revision as of 17:04, 7 June 2017
Line 25:		Line 25:
	##For standards, add 0-5 and .5ul of glycerol standard		##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.		##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.		##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.		##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.

Pfeiferl at 17:04, 7 June 2017

2017-06-07T17:04:27Z

← Older revision		Revision as of 17:04, 7 June 2017
Line 25:		Line 25:
	##For standards, add 0-5 and .5ul of glycerol standard		##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.		##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.		##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.

Mollyec: /* Protocol */

2017-05-16T15:17:51Z

Protocol

Davebrid: Undo revision 853 by Davebridges (talk)

2014-04-29T12:30:37Z

Undo revision 853 by Davebridges (talk)

← Older revision		Revision as of 12:30, 29 April 2014
Line 8:		Line 8:
	==Protocol==		==Protocol==
	#Weigh out 30mg tissue (record weights for normalization) on dry ice into round bottom eppendorf tube. Add one stainless steel ball bearing.		#Weigh out 30mg tissue (record weights for normalization) on dry ice into round bottom eppendorf tube. Add one stainless steel ball bearing.
	#Add ~~200ul~~ Homogenization Buffer		#Add 500ul Homogenization Buffer
	#Homogenize ~~by sonicating on ice~~ 3 ~~x 15s~~ or ~~3 x freeze thaws.~~		#Homogenize with Qiagen Tissue Lyser for 3 minutes @ 25Hz for Liver/WAT or for 5 minutes @ 30Hz for muscle
	#Add ~~5 ul~~ KOH		#Add 12.5ul KOH
	#Mix by inverting		#Mix by inverting
	#Add 800ul '''Chloroform/Methanol Mixture'''		#Add 800ul '''Chloroform/Methanol Mixture'''

Davebrid: Updated protocol for tissue culture

2014-04-29T12:30:05Z

Updated protocol for tissue culture

← Older revision		Revision as of 12:30, 29 April 2014
Line 8:		Line 8:
	==Protocol==		==Protocol==
	#Weigh out 30mg tissue (record weights for normalization) on dry ice into round bottom eppendorf tube. Add one stainless steel ball bearing.		#Weigh out 30mg tissue (record weights for normalization) on dry ice into round bottom eppendorf tube. Add one stainless steel ball bearing.
	#Add ~~500ul~~ Homogenization Buffer		#Add 200ul Homogenization Buffer
	#Homogenize ~~with Qiagen Tissue Lyser for~~ 3 ~~minutes @ 25Hz for Liver/WAT~~ or ~~for 5 minutes @ 30Hz for muscle~~		#Homogenize by sonicating on ice 3 x 15s or 3 x freeze thaws.
	#Add ~~12.5ul~~ KOH		#Add 5 ul KOH
	#Mix by inverting		#Mix by inverting
	#Add 800ul '''Chloroform/Methanol Mixture'''		#Add 800ul '''Chloroform/Methanol Mixture'''

Mpeloqu1: /* Protocol */

2013-07-31T21:26:30Z

Protocol

← Older revision		Revision as of 21:26, 31 July 2013
Line 7:		Line 7:

	==Protocol==		==Protocol==
	#~~Use 5 female or 8 Male flies~~ for ~~Assay~~		#Weigh out 30mg tissue (record weights for normalization) on dry ice into round bottom eppendorf tube. 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
	#~~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 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.
			##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 standars, add 0-5ul of glycerol standard
			##Add 5ul of resuspended lipid to each well to start (also make a 5ul blank of the butanol mixture) and mix.
			##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.

Mpeloqu1: /* Protocol */ Creation of Protocol

2013-07-31T21:11:21Z

Protocol: Creation of Protocol

← Older revision		Revision as of 21:11, 31 July 2013
Line 7:		Line 7:

	==Protocol==		==Protocol==
	# ~~Weigh out 30 mg tissue (record weight~~ for ~~normalization) on dry ice into round bottom eppendorf tube. Add one stainless steel ball bearing.~~		#Use 5 female or 8 Male flies for Assay
	~~# Add 500 uL Homogenization Buffer.~~		#Homogenize flies (3 min @ 40Hz) in 1mL of .05% Tween (from Tween-20 stock)
	# Homogenize ~~with qiagen tissue lyser~~ 3 ~~minutes at 25 Hz for Liver/WAT or 5~~ min ~~at 30 Hz for muscle~~.		#Heat samples in 70C waterbath for 5 minutes
	# ~~Add 12.~~5 ~~uL KOH~~		#Spin samples @ 5000G for 1 minute
	# ~~Mix by inverting~~		#Transfer 500ul of supernatent in new microfuge tube
	~~# Add 800 uL '''Chloroform/Methanol Mixture'''~~		#Spin @ 14000G for 3 minutes
	~~# Vortex vigorously then sit at room temperature~~ for ~~5 min~~		#Add 50ul of sample to 200ul of TG solution in a 96 well plate
	# ~~Centrifuge 10min at 13 000 RPM~~		#Incubate plate @ 37C for 5 mins
	~~# Take the bottom layer into a fresh labelled~~ tube.		#Measure 540nm absorbance
	# ~~Dry in fume hood overnight (or until completely dry)~~
	~~# If absorbance is going to be measured by cuvette, use non-bolded values. If you are using a plate reader use bolded values.~~
	# Add ~~500uL '''(50uL)''' of '''Butanol Mixture'''. See [[#Suggested Volumes \| Suggested Volumes]] for your specific tissue~~
	~~# Measure triglyceride levels using Sigma Diagnostic Kit using 5 uL~~ of sample:
	~~## Resuspend triglyceride and glycerol reagent with water if necessary.~~
	~~## Calculate how many samples you have (samples + buffer blank + 6 standard curve values).~~
	~~## 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.~~
	~~## Aliquot 700 uL into a cuvette or '''100 uL into a well of~~ a 96 well plate~~'''.~~
	#~~# For standards add 0-5 uL of glycerol standard.~~
	~~## Add 5 uL of resuspended lipid to each well to start (also make a 5uL blank of the butanol mixture) and mix.~~
	~~## 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.~~
	## Measure absorbance ~~at 540 nm.~~
	~~## If any samples are A540<0.1 or above the 5uL standard A540 then repeat with more or less lipid as required.~~

← Older revision		Revision as of 15:17, 16 May 2017
Line 7:		Line 7:

	==Protocol==		==Protocol==
	#Weigh out ~~30mg~~ tissue (record weights for normalization) on dry ice into round bottom eppendorf tube. Add one stainless steel ball bearing.		#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.
	#Add 500ul Homogenization Buffer		#Add 500ul Homogenization Buffer
	#Homogenize with Qiagen Tissue Lyser for 3 minutes @ 25Hz for Liver/WAT or for 5 minutes @ 30Hz for muscle		#Homogenize with Qiagen Tissue Lyser for 3 minutes @ 25Hz for Liver/WAT or for 5 minutes @ 30Hz for muscle
	#Add 12.5ul KOH		#Add 12.5ul KOH
	#Mix by inverting		#Mix by inverting then transfewr sample to a new 1.5 mL tube. Place dirty ball bearing in ethanol (located in the fume hood)
	#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 for 10 minutes @ 13000G
	#Transfer the bottom layer into a new tube		#Transfer 400 ul of 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.~~		#Add'''(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.
	##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 '''100ul into a well of a 96 well plate'''
	##For ~~standars~~, add 0-5ul of glycerol standard		##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.		##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.