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)

     1. • 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

• 1. At exactly 30 minutes post insulin stimulation, in the hot room, aspirate the media and wash cells with ice cold 1x PBS 3 times.

  2. Lyse the cells in 0.5 mL/well of 0.1% SDS, rock for 30 minutes

  3. Transfer to eppendorf tubes and vortex well

     • At this point they can be stored at -4 ̊C until ready to use.

  4. Take 300 μL of each in scintillation vials containing 4 mL fluid & mix well

  5. Normalize to protein (BCA Protein Assay)