Glucose Uptake Assay
Revision as of 20:42, 18 February 2013 by Davebridges (Talk | contribs) (specified insulin concentration)
Revision as of 20:42, 18 February 2013 by Davebridges (Talk | contribs) (specified insulin concentration)
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.
- Insulin stock solution (1 mg/mL)
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 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)