Glucose Uptake Assay: Difference between revisions
specified insulin concentration |
→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 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 | *'''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== | ||