Procedure

Materials required

1. 12 well microtiter plate

2. 0.1 M Krebs-Ringer-Phosphate (KRP) buffer (pH: - 7.2- 7.4)

3. 0.1% SDS

4. Serum free DMEM

5. Insulin(100nM) & Plant extracts(100µg/ml)

6. Radioactive cocktail (10µM 2-deoxy glucose and 0.25µCi of 2 deoxy-D-(3H) glucose in 12 ml of KRP buffer)

7. 1.5 ml Eppendorf tube

8. 15 ml centrifuge tubes

9. Liquid Scintillation Counter

10. Pipettes

Procedure

1. Take out the differentiated 3T3 L1 cell plate from CO2 incubator.

2. Remove the medium from the 12 well plate using a suction pump.

3. Add 1000 serum free media into all wells of the 12 well plate.

4. Serum starves the cells for 2 hours in CO2 incubator.

5. Remove the media using a suction pump.

6. Add Basal to the wells 1A, 1B and 1C.

7. Add Insulin to the wells 2A, 2B and 2C.

8. Add plant extract X and Y to 3A, 3B, 3C, 4A, 4B and 4C respectively.

9. Incubate the plate at 370C for 30 minutes.

10. Take out the plate and remove the media.

11. Add 1000 KRP buffer (in room temperature) into all wells and wash cells.

12. Remove the KRP buffer completely.

13. Remove the trace amount of KRP buffer also using a pipette.

14. Add 1000 µl radioactive cocktail into each well.

15. Incubate the plate for 5 minutes in a CO2 incubator.

16. Discard the cocktail and wash twice with 1000µl ice cold KRP buffer.

17. Add 1000µl of 0.1% SDS to all wells to lyse the cells.

18. Then scrap in each well using a cell scraper.

19. Take out the whole cell lysate from each well and add to corresponding labelled scintillation tubes contains 4 ml scintillation fluid.

20. Mix the tubes gently and keep it for 1-2 minutes to settle all the fluids.

21. Then measure the glucose uptake rate using the Liquid Scintillation counter.