Difference between revisions of "Glucose Uptake Assay"
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==Materials== | ==Materials== | ||
| − | *2-Deoxyglucose (cold): Sigma D-6134. Dissolve 32.8mg/mL (200 mM) in KRBH | + | *'''Cold 2-DG.''' 2-Deoxyglucose (cold): Sigma D-6134. Dissolve 32.8mg/mL (200 mM) in [[KRBH Buffer]] |
| − | *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. 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 | + | *'''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== | ==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 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 | + | #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 [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: Glucose Uptake]] | ||
| + | [[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
- 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 PBS per well
- Scrape cells with an upside down p200 tip
- Add 400 uL cells to 5 mL aqueous scintillation fluid, vortex and count on program 3
- Do a bradford assay on 50 uL of cells (use PBS as blank)
