Glucose Uptake Assay: Difference between revisions
added templates |
No edit summary |
||
| Line 3: | Line 3: | ||
*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 | ||
*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 3X5 ul of hot 2DG. | ||
==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 (100nM) 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 | ||
| Line 17: | Line 17: | ||
#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== | ==Analysis== | ||