REAGENTS:
KRH for 1L
50mM HEPES, pH 7.4 5.2g (or 20mL of 1M stock)
136mM NaCl 7.9g (or 27.2 ml of 5 stock)
4.7mM KCl 0.35g (or 4.7 ml of 1M stock)
1.25mM MgSO4 0.3g (or 1.25 ml of 1M stock)
1.25mM CaCl2 0.5ml of 2.5 M stock
1mM 2-deoxyglucose (0.164g/10ml=0.1M)
5mCi/ml [3H]-2-deoxyglucose (Dupont NEN:2-[1,2-3H]-deoxy-D-glucose)
final concentration START on cells: 100mM 2-deoxyglucose,0.5mCi/ml [3H]-2-deoxyglucose
Cytochalasin B (Sigma): 25mM in EtOH
PROTOCOL:
1. Wash cells 2x in DMEM/0.1%BSA at 37°C
2. Do serum-free stepdown for 4 hours in DMEM/BSA
3. Wash cells 2x KRH/0.1% BSA, 37°C
4. Do glucose-free stepdown in 0.45ml KRH/0.1% BSA, 37°C
5.Add cytochalasin B to final 10mM at time of stimulation for non-specific uptake
6. Last 5 minutes of insulin stimulation add 50ml of 10x START
7.At the end of incubation wash cells in 12-well plates by dunking into large beaker of ice-cold PBS. Put plates in ice and wash 2x in ice-cold PBS
8. Solubilize cells in 0.5ml 0.05% SDS
9. Count 0.4ml in scintillation fluid and count 50ml of 10X START for specific activity calculations
10. Save remaining lysates for protein determination. Plates can be stored by wrapping in Parafilm and storing at 4°C
11. Calculate specific activity of glucose uptake by doing protein assays and subtracting the cytoB cpm values from all wells. Express as xmol/mg/min
Example calculation:
--(0.1mmol/L glucose)(0.0005L)(0.4ml/0.5ml)=0.00004mmol=0.04mmol 2-deoxyglucose counted
--if 50ml of START=300,000cpm, then one should use 300,000 cpm (0.4/0.5)=240,000 (since only 4/5 of well is counted)
--240,000 cpm/0.04 mmole=6 x 106 cpm/mmol
---Then, for example, 6000cpm (after subtracting cyto B value) /6 x 106 cpm/mmol=0.001mmol=1nmole of 2-deoxyglucose uptake
--assuming [prot]/well=0.2 mg and time of uptake= 4minutes, 1then 1nmol/0.2mg/4min=1.25 nmol/mg/min