I am having problems obtaining insulin stimulated glucose uptake in differentiated 3T3-L1 adipocytes. I have tried several differentiation protocols but I only get a 30% increase in glucose uptake after insulin stimulation. The protocol for [14C]-2-deoxy-D-glucose uptake I follow are from J.M Olefsky's group, in particular this paper: https://www.sciencedirect.com/science/article/pii/S0021925819482094

I do get differentiated 3T3-L1 adipocytes, using several different differentiation protocols. But for some reason the cells barely respond to insulin. I wonder about a few things that might cause my problem.

1. At what time point the cells are best suitable for glucose uptake experiments? Different papers write about 10-12 days post-differentiation, others talk about 10-12 days post differentiation initiation. since it takes a while before the cells are fully differentiated there is a difference between post differentiation and post differentiation initiation.

2. How long should the cells be serum starved and glucose starved?

3. Should DMEM with high or low glucose be used and with or without pyruvate?

I hope someone can help, because we have no idea what causes the lack of insulin stimulated glucose uptake in the cells.