3H-2-DG assay

Glucose Uptake Assay

Solutions needed :

1. KRH Buffer. pH is critical. Warm up at 37 C before starting the experiment. 

2. KRH + Insulin (Final Concentration: 100nM of insulin in KRH).

3. Washing Buffer

200mM glucose in KRH buffer. (Add 10 ml 2M glucose solution and 90ml KRH Buffer). 

4. Lysis buffer : 0.1% SDS Solution

5. 4 ml each of Ecolite solution in scintillation vials. 

6. 2DG (2-Deoxy-D-glucose, 98%, FW 164.16), working solution: 100mM 2DG in water(16.416mg/ml)

7. Radioisotope : 2-deoxy-d-[2,6-3H]-glucose (0.33 mCi/ml)

  * from PerkinElmer NEN Radiochemicals

Protocol:

1. Wash cells with 250ul Serum free DMEM once, then add 200ul serum free DMEM 

2. Place in incubator(37C) for 6 hours. 

3. Wash cells with 250ul KRH Buffer once. 

4. Stimulation for 20 minutes in incubator(37C) after adding the following accordingly 

- Basal: Add 200ul KRH Buffer

- Insulin: Add 200ul 100nM Insulin in KRH Buffer

5. On the stimulation Buffer add 25 ul per well of radioisotope+2DG+KRH Buffer for both Basal and Insulin. 

6. Place in incubator (37C) for 10 mins 

7. Wash 3 times with 280ul per well of washing buffer which is KRH + 200mM Glucose  

9. Lysis cells with 200ul of 0.1% SDS for 20 minutes. 

10. Collect cells by pipetting up and down and add to scintillation vials which contain 4ml Ecolite solution. Save a small volume of lysates for protein quantification.

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