I've been trying to induce knock-down in differentiated 3T3-L1 using DeliverX plus siRNA reagent.
In this protocol, I have to detach the differnentiated cells 24h before transfection. and for me, it's hard to reattach the differentiated cells it that more differentiated cells hardly attach to the culture dishes. And anyhow, through the several trials, i can find out sub-optimal(not optimal) condition to reattach these cells.
1. Does anyone know optimal condition to detach and reattach the cells(differentiation state, cell numbers etc)?
2. In paper, people can induce a huge increase in insulin-induced glucose uptake in these cells, but i cannot induce any response when i use 20nM insulin. Of course, i can see insulin-dependent signaling by western blotting.
If anyone can give me comments about these, i'd be great!