I am trying to use Percoll gradients to enrich for progenitor cells isolated from organs. The cells can be dissociated and are viable afterwards. I am using Percoll Plus from GE Healthcare. I have been using the protocol in the attached link. If I follow the protocol exactly, I am able to isolate cells at the different density interfaces, however, all the cells are dead when counted using trypan blue exclusion and a hemocytometer. I found some other protocols that use 400 x g for centrifugation and I have set different ranges of RCF from 200 to 600 times g. In every case, the cells have been permeable to trypan blue. I will try a ficoll underlay and I am thinking about Nicodenz. Has anyone else had this issue? How did you overcome it? Any feedback is welcome. TIA.