Difference between revisions of "Glucose Uptake Assay"

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(Materials: updated with catalog numbers)
 
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==Materials==
 
==Materials==
*2-Deoxyglucose (cold): Sigma D-6134.  Dissolve 32.8mg/mL (200 mM) in KRBH
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*'''Cold 2-DG.'''  2-Deoxyglucose (cold): Sigma D-6134.  Dissolve 32.8mg/mL (200 mM) in [[KRBH Buffer]]
*0.5% BSA in KRBH
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*0.5% BSA in [[KRBH Buffer]]
*Radioactive <sup>14</sup>C-2-deoxyglucose
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*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
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*'''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.
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*Insulin stock solution (1 mg/mL) if doing insulin stimulation
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*Scintillation Fluid
  
 
==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)
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#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
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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)
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#Do a bradford assay on 50 uL of cells (use PBS as blank)
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==Analysis==
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*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]
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*For sweave use this template found on Github [https://github.com/davebridges/biomolecule-scripts/blob/master/R/Sweave/2-dg%20analysis.Rnw]
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[[Category: Metabolic Measurements]]
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[[Category: Glucose Uptake]]
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[[Category: Cell Culture]]

Latest revision as of 14:55, 31 July 2018

Materials

  • Cold 2-DG. 2-Deoxyglucose (cold): Sigma D-6134. Dissolve 32.8mg/mL (200 mM) in KRBH Buffer
  • 0.5% BSA in KRBH Buffer
  • Radioactive 14C-2-deoxyglucose. DEOXY-D-GLUCOSE, 2-[14C(U)], order from American Radiolabeled Chemical, 50 uCi is enough for about 50 experiments. (catalog 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 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) if doing insulin stimulation
  • Scintillation Fluid

Protocol

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

Analysis

  • For excel fill in orange cells in this template found on Github [1]
  • For sweave use this template found on Github [2]