Hello everyone. I am curently working on a protocol to measure glucose uptake in HepG2 cells using 2-NDBG. The work involves the pre-treatment of cells with an active material and observe if it has an effect on increasing glucose uptake. Metformin is used as a positive control. I have come across several threads on ReaserchGate which gave me the impression that this experiment is kind of tricky.

I have read several references. In some references, the cells are pre-treated with the drug, next incubated with glucose-free medium, and lastly the fluorescent 2-NBDG (in glucose-free medium) is added. In others, after incubation with the drug and before the addition of 2-NBDG, there is an extra step in which cells are stimulated with insulin for only 15 min.

I have a couple of questions.

-Is the stimulation with insulin necessary? Or I can skip this step?

- What´s the mechanism that makes metformin or other drug pre-treated with cells and then completely washed away cause an increase in glucose uptake? Why the drug is not added simultanously with 2-NBDG?

-Why after treatment cells are incubated with glucose-free medium? How important is this step? What happens if we drop this step and incubate cells directly with 2-NBDG?

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