*Do following in the hood*
- Differentiate the 3T3-L1s to >day 10
- Wash 1x with cells with 1xKRH media
- Serum starve the cells in 0.5% BSA/KRH for >2 hours
- Wash the cells with KRH
- Add 0.5 mL of 0.5% BSA/KRH (exact amount)
- Monitor meticulous timing for the next steps
- 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*
- Cytocholasin B, 1 uL/well: 10 minutes
- [Final]: 50 μM, [Stock]: 25 mM *Gently swirl and rock cells*
- 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 Reagent | Per Well | Per 70 Wells | | Cold DOG | 0.25 µL | 17.5 µL | | Hot DOG | 1.00 µL | 70 µL | | KRH | 3.75 µL | 262.5 µL | | Final Volume | 5.0 µL | 350 µL |
- At exactly 30 minutes post insulin stimulation, in the hot room, aspirate the media and wash cells with ice cold 1x PBS 3 times.
- Lyse the cells in 0.5 mL/well of 0.1% SDS, rock for 30 minutes
- Transfer to eppendorf tubes and vortex well
- At this point they can be stored at -4 ̊C until ready to use.
- Take 300 μL of each in scintillation vials containing 4 mL fluid & mix well
- Normalize to protein (BCA Protein Assay)
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