Glucose Uptake Assay

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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]