I want to do an ELISA on HUVEC cell culture supernatant to check for TNF alpha release. Can anybody recommend a suitable cell density/ml medium? I am thinking to culture my cells in a 6 well plate, but I am scared to dilute the cytokines too much. My boss recommended to culture the cells in 500µl/6-well, but I don't think that this will work, if I want to keep the culture for 48h. Thanks a lot!