what is the best blood tubes for PBMC isolation, between EDTA,...

Temperature is critical as it changes the density of your gradient. Gradients are meant to be used at room temperature. Using them at 4°C could result in higher contamination with unwanted cell types such as granulocytes. Higher speeds of centrifugation are fine when you are using fresh healthy cells. However, if you are using frozen PBMC, then you should use slower speeds during your washes when you thaw them because they are more fragile. I believe I used 300 g for thawed PBMC. The ratio of Ficol to blood isn't your problem since you don't really mix the two. The problem is the density of your plasma vs the density of the gradient. The bigger the difference, the tighter the band, which is why dilution of the blood is helpful. If your temperature is low, then the Ficol will become more dense and you will lose the separation of your granulocytes and lymphocytes. As mentioned before, you can do a gentle spin to get the plasma and then dilute the blood to do the layering onto Ficol.

thank you for your answer!

RT and 4C doesn't matter but you should use 800-850 g.

Heparinised tube is good.