Difference between revisions of "Glucose Uptake Assay"

Revision as of 20:42, 18 February 2013

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

1. Starve cells >3h in 0.5% FBS
2. Prepare insulin in KRBH/BSA (0.6uL insulin/mL), needing 0.5 mL per well)
3. Wash cells 2x with warm PBS -/-
4. Add 450 uL KRBH/BSA with or without insulin to wells. Typically do triplicate measurements
5. Wait 30 min
6. Add 50 uL hot 2-DG solution per well and start timer
7. After 5 min add 50 uL cold 2-DG
8. Wash cells 3x1mL with cold PBS
9. Add 500 uL PBS per well
10. Scrape cells with an upside down p200 tip
11. Add 400 uL cells to 5 mL aqueous scintillation fluid, vortex and count on program 3
12. Do a bradford assay on 50 uL of cells (use PBS as blank)

Analysis

• For excel fill in orange cells in this template found on Github [1]
• For sweave use this template found on Github [2]