Radioactive 14C-2-deoxyglucose. DEOXY-D-GLUCOSE, 2-[14C(U)], order from American Radiolabeled Chemical, 50 uCi is enough for about 50 experiments. (catalog ARC 0112A-50 µCi)
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 3 x 5 uL of hot 2DG to determine counts per pmol of 2-DG. This volume is enough for 20 wells, so you can scale up or down if needed.
Insulin stock solution (1 mg/mL) if doing insulin stimulation
Scintillation Fluid
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)
Analysis
For excel fill in orange cells in this template found on Github [1]