Glucose Uptake Assay - Revision history

Davebrid: /* Materials */ updated with catalog numbers

2018-07-31T14:55:03Z

Materials: updated with catalog numbers

← Older revision		Revision as of 14:55, 31 July 2018
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	==Materials==		==Materials==
	*2-Deoxyglucose (cold): Sigma D-6134. Dissolve 32.8mg/mL (200 mM) in [[KRBH Buffer]]		*'''Cold 2-DG.''' 2-Deoxyglucose (cold): Sigma D-6134. Dissolve 32.8mg/mL (200 mM) in [[KRBH Buffer]]
	*0.5% BSA in [[KRBH Buffer]]		*0.5% BSA in [[KRBH Buffer]]
	*Radioactive <sup>14</sup>C-2-deoxyglucose		*Radioactive <sup>14</sup>C-2-deoxyglucose. DEOXY-D-GLUCOSE, 2-[14C(U)], order from American Radiolabeled Chemical, 50 uCi is enough for about 50 experiments. (catalog [https://www.arcincusa.com/featured-products-m/14c-labeled-compounds/Deoxy-D-glucose-2-14C-U-ARC-0112A-50-%C2%B5Ci-detail ARC 0112A-50 µCi])
	*Hot 2-DG. Need 50 uL per well, prepare 1 mL KRBH/BSA, 1 uL cold 2-DG and 50 uL radioactive 2-DG. count ~~3X5 ul~~ of hot 2DG.		*'''Hot 2-DG'''. Need 50 uL per well, prepare 1 mL KRBH/BSA, 1 uL cold 2-DG and 50 uL radioactive 2-DG. count 3 x 5 uL of hot 2DG to determine counts per pmol of 2-DG. This volume is enough for 20 wells, so you can scale up or down if needed.
	*Insulin stock solution (1 mg/mL)		*Insulin stock solution (1 mg/mL) if doing insulin stimulation
			*Scintillation Fluid

	==Protocol==		==Protocol==

Davebrid: specified insulin concentration

2013-02-18T20:42:44Z

specified insulin concentration

← Older revision		Revision as of 20:42, 18 February 2013
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	*Radioactive <sup>14</sup>C-2-deoxyglucose		*Radioactive <sup>14</sup>C-2-deoxyglucose
	*Hot 2-DG. Need 50 uL per well, prepare 1 mL KRBH/BSA, 1 uL cold 2-DG and 50 uL radioactive 2-DG. count 3X5 ul of hot 2DG.		*Hot 2-DG. Need 50 uL per well, prepare 1 mL KRBH/BSA, 1 uL cold 2-DG and 50 uL radioactive 2-DG. count 3X5 ul of hot 2DG.
			*Insulin stock solution (1 mg/mL)

	==Protocol==		==Protocol==
	#Starve cells >3h in 0.5% FBS		#Starve cells >3h in 0.5% FBS
	#Prepare insulin in KRBH/BSA (0.6uL ~~(100nM)~~ insulin/mL, needing 0.5 mL per well)		#Prepare insulin in KRBH/BSA (0.6uL insulin/mL), needing 0.5 mL per well)
	#Wash cells 2x with warm PBS -/-		#Wash cells 2x with warm PBS -/-
	#Add 450 uL KRBH/BSA with or without insulin to wells. Typically do triplicate measurements		#Add 450 uL KRBH/BSA with or without insulin to wells. Typically do triplicate measurements

Irith at 21:03, 11 July 2012

2012-07-11T21:03:35Z

← Older revision		Revision as of 21:03, 11 July 2012
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	*0.5% BSA in [[KRBH Buffer]]		*0.5% BSA in [[KRBH Buffer]]
	*Radioactive <sup>14</sup>C-2-deoxyglucose		*Radioactive <sup>14</sup>C-2-deoxyglucose
	*Hot 2-DG. Need 50 uL per well, prepare 1 mL KRBH/BSA, 1 uL cold 2-DG and 50 uL radioactive 2-DG		*Hot 2-DG. Need 50 uL per well, prepare 1 mL KRBH/BSA, 1 uL cold 2-DG and 50 uL radioactive 2-DG. count 3X5 ul of hot 2DG.

	==Protocol==		==Protocol==
	#Starve cells >3h in 0.5% FBS		#Starve cells >3h in 0.5% FBS
	#Prepare insulin in KRBH/BSA (0.6uL insulin/mL, needing 0.5 mL per well)		#Prepare insulin in KRBH/BSA (0.6uL (100nM) insulin/mL, needing 0.5 mL per well)
	#Wash cells 2x with warm PBS -/-		#Wash cells 2x with warm PBS -/-
	#Add 450 uL KRBH/BSA with or without insulin to wells. Typically do triplicate measurements		#Add 450 uL KRBH/BSA with or without insulin to wells. Typically do triplicate measurements
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	#Scrape cells with an upside down p200 tip		#Scrape cells with an upside down p200 tip
	#Add 400 uL cells to 5 mL aqueous scintillation fluid, vortex and count on program 3		#Add 400 uL cells to 5 mL aqueous scintillation fluid, vortex and count on program 3
	#Do a bradford assay on 20 uL of cells (use PBS as blank)		#Do a bradford assay on 50 uL of cells (use PBS as blank)

	==Analysis==		==Analysis==

Davebrid: added templates

2011-11-03T13:44:38Z

added templates

← Older revision		Revision as of 13:44, 3 November 2011
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	#Add 400 uL cells to 5 mL aqueous scintillation fluid, vortex and count on program 3		#Add 400 uL cells to 5 mL aqueous scintillation fluid, vortex and count on program 3
	#Do a bradford assay on 20 uL of cells (use PBS as blank)		#Do a bradford assay on 20 uL of cells (use PBS as blank)

			==Analysis==
			*For excel fill in orange cells in this template found on Github [https://github.com/davebridges/biomolecule-scripts/blob/master/Excel%20Templates/2-DG%20Template.xlsx]
			*For sweave use this template found on Github [https://github.com/davebridges/biomolecule-scripts/blob/master/R/Sweave/2-dg%20analysis.Rnw]

	[[Category: Metabolic Measurements]]		[[Category: Metabolic Measurements]]
	[[Category: Glucose Uptake]]		[[Category: Glucose Uptake]]
	[[Category: Cell Culture]]		[[Category: Cell Culture]]

Davebrid: added categories

2011-01-12T19:46:45Z

added categories

← Older revision		Revision as of 19:46, 12 January 2011
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	#Add 400 uL cells to 5 mL aqueous scintillation fluid, vortex and count on program 3		#Add 400 uL cells to 5 mL aqueous scintillation fluid, vortex and count on program 3
	#Do a bradford assay on 20 uL of cells (use PBS as blank)		#Do a bradford assay on 20 uL of cells (use PBS as blank)

			[[Category: Metabolic Measurements]]
			[[Category: Glucose Uptake]]
			[[Category: Cell Culture]]

Davebrid at 18:30, 14 October 2009

2009-10-14T18:30:41Z

← Older revision		Revision as of 18:30, 14 October 2009
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	==Materials==		==Materials==
	*2-Deoxyglucose (cold): Sigma D-6134. Dissolve 32.8mg/mL (200 mM) in KRBH		*2-Deoxyglucose (cold): Sigma D-6134. Dissolve 32.8mg/mL (200 mM) in [[KRBH Buffer]]
	*0.5% BSA in [[KRBH Buffer]]		*0.5% BSA in [[KRBH Buffer]]
	*Radioactive <sup>14</sup>C-2-deoxyglucose		*Radioactive <sup>14</sup>C-2-deoxyglucose

Davebrid: added link to KRBH Buffer

2009-10-14T18:19:24Z

added link to KRBH Buffer

← Older revision		Revision as of 18:19, 14 October 2009
Line 1:		Line 1:
	==Materials==		==Materials==
	*2-Deoxyglucose (cold): Sigma D-6134. Dissolve 32.8mg/mL (200 mM) in KRBH		*2-Deoxyglucose (cold): Sigma D-6134. Dissolve 32.8mg/mL (200 mM) in KRBH
	*0.5% BSA in KRBH		*0.5% BSA in [[KRBH Buffer]]
	*Radioactive <sup>14</sup>C-2-deoxyglucose		*Radioactive <sup>14</sup>C-2-deoxyglucose
	*Hot 2-DG. Need 50 uL per well, prepare 1 mL KRBH/BSA, 1 uL cold 2-DG and 50 uL radioactive 2-DG		*Hot 2-DG. Need 50 uL per well, prepare 1 mL KRBH/BSA, 1 uL cold 2-DG and 50 uL radioactive 2-DG

Davebrid at 00:13, 27 May 2009

2009-05-27T00:13:56Z

← Older revision		Revision as of 00:13, 27 May 2009
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	#After 5 min add 50 uL cold 2-DG		#After 5 min add 50 uL cold 2-DG
	#Wash cells 3x1mL with cold PBS		#Wash cells 3x1mL with cold PBS
	#Add 500 uL per well		#Add 500 uL PBS per well
	#Scrape cells with a p200 tip		#Scrape cells with an upside down p200 tip
	#Add 400 uL cells to 5 mL aqueous scintillation fluid, vortex and count on program 3		#Add 400 uL cells to 5 mL aqueous scintillation fluid, vortex and count on program 3
	#Do a bradford assay on 20 uL of cells (use PBS as blank)		#Do a bradford assay on 20 uL of cells (use PBS as blank)

Davebrid: initial page

2009-05-26T21:37:15Z

initial page

New page

==Materials==
*2-Deoxyglucose (cold): Sigma D-6134. Dissolve 32.8mg/mL (200 mM) in KRBH
*0.5% BSA in KRBH
*Radioactive <sup>14</sup>C-2-deoxyglucose
*Hot 2-DG. Need 50 uL per well, prepare 1 mL KRBH/BSA, 1 uL cold 2-DG and 50 uL radioactive 2-DG

==Protocol==
#Starve cells >3h in 0.5% FBS
#Prepare insulin in KRBH/BSA (0.6uL insulin/mL, needing 0.5 mL per well)
#Wash cells 2x with warm PBS -/-
#Add 450 uL KRBH/BSA with or without insulin to wells. Typically do triplicate measurements
#Wait 30 min
#Add 50 uL hot 2-DG solution per well and start timer
#After 5 min add 50 uL cold 2-DG
#Wash cells 3x1mL with cold PBS
#Add 500 uL per well
#Scrape cells with a p200 tip
#Add 400 uL cells to 5 mL aqueous scintillation fluid, vortex and count on program 3
#Do a bradford assay on 20 uL of cells (use PBS as blank)