Glucose Uptake Assay: Difference between revisions
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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 | *'''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: Metabolic Measurements]] | ||
[[Category: Glucose Uptake]] | [[Category: Glucose Uptake]] | ||
[[Category: Cell Culture]] | [[Category: Cell Culture]] | ||