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==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)
*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)
