2. Allow the cells to attain 90-95% confluency
3. Replace the medium with 1 ml of DMEM with 2% serum per well
4. Allow the cells to differentiate for 4 days
5. Change the medium to DMEM without serum
6. Treat L6 myotubes with various dilutions of the plant extract
7. Incubate the plate for 24 hrs at 37'C in 5% CO2
8. After incubation, discard the medium
9. Add 1 ml of KRPH buffer per well and pulse it with 0.5 uCi per well (5 ul/ml) of 2- Deoxy D3[H] Glucose
10. Incubate for 20 min in 5% CO2 at 37'C
11. Discard the buffer and terminate the glucose uptake by adding 1 ml of ice-cold KRPH buffer per well
12. Discard the content and add 500 ul of STV
13. Scrape the cells and transfer it to 1.5 ml microfuge tubes
14. Centrifuge at 800 rpm for 7 min
15. Discard the supernatant and suspend the pellet in 1 ml of KRPH buffer
16. Centrifuge at 8000 rpm for 7 min
17. Discard the supernatant
18. Lyse the pellet with 400 ul of 0.1% SDS
19. Mix the lysate thoroughly
20. Incubate the lysate at Room Temperature overnight
21. Transfer 200 ul of the lysate to the scintillation vials containing glass filter paper
22. Allow it to dry overnight
23. Add 5 ml of scintillation fluid to each vial
24. Take count using HTS
25. Perform the assay in triplicates to get concordant values
26. Plot the graph with drug dilutions vs. CPM
27. Use control, solvent control and positive control for efficient evaluation of the assay