Difference between revisions of "Glucose Uptake Assay"
From Bridges Lab Protocols
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*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== | ||
Revision as of 21:03, 11 July 2012
Materials
- 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
- 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
- Starve cells >3h in 0.5% FBS
- Prepare insulin in KRBH/BSA (0.6uL (100nM) 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)
