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Insulin Stimulated Glucose Uptake Assay

*Do following in the hood*
  1. Differentiate the 3T3-L1s to >day 10
  2. Wash 1x with cells with 1xKRH media
  3. Serum starve the cells in 0.5% BSA/KRH for >2 hours
  4. Wash the cells with KRH
  5. Add 0.5 mL of 0.5% BSA/KRH (exact amount)
    • Monitor meticulous timing for the next steps
  6. Insulin, 5μL/well: 10 minutes      **FAST STEPS** Use electronic pipettor
    • [Final]: 10 nM (make 5.73μL of our stock to 1 mL of KRH to give 1 μM stock), Insulin ([stock]= 1 μM; [working]= 10 nM) in KRH. 1 μM stock- 5 μg/mL
    • The insulin that we use for tissue culture is 1 mg/mL, which is 174.4 μM. Add 5.73 μL of this to 1 mL of KRH to get 1 μM of insulin stock. For 0.2μM stock, dilute the 1 μM stock to 1:5 in KRH     *Gently swirl and rock cells*
  7. Cytocholasin B, 1 uL/well: 10 minutes
    • [Final]: 50 μM, [Stock]: 25 mM  *Gently swirl and rock cells*
  8. DOG (2-deoxy-D[2-14C(U)], 5 μL/well: 5 minutes
    • Use electronic pipettor
    • [Final]: 0.1uCi/mL in 500 μl buffer [Stock]: 25 mM
    • Cold glucose concentration= 200 mM in KRH, 0.036 g/1 mL KRH

      Hot ReagentPer WellPer 70 Wells
      Cold DOG0.25 µL17.5 µL
      Hot DOG1.00 µL70 µL
      KRH 3.75 µL 262.5 µL 
      Final Volume 5.0 µL 350 µL 

  9. At exactly 30 minutes post insulin stimulation, in the hot room, aspirate the media and wash cells with ice cold 1x PBS 3 times.
  10. Lyse the cells in 0.5 mL/well of 0.1% SDS, rock for 30 minutes
  11. Transfer to eppendorf tubes and vortex well
    • At this point they can be stored at -4 ̊C until ready to use.
  12. Take 300 μL of each in scintillation vials containing 4 mL fluid & mix well
  13. Normalize to protein (BCA Protein Assay)