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.
Here is the link for the detailed protocol. https://www.encodeproject.org/documents/f27f883a-17af-461e-b05e-3af8c41703d6/@@download/attachment/Cell_purification_using_Percoll_V1.pdf
Matthew,
Is it possible that the osmotic strength of the Percoll gradient is "off".
Good question. I buy the Percoll Plus from GE healthcare. I make up the solution using the ENCODE protocol with 10x PBS supplemented with 1M HEPES pH 7.2. What variables would you change to adjust the osmotic pressure, a gradient of NaCl? I have been thinking about using 10x DMEM to make the layers.
Hi,
We use 10x PBS without HEPES to make up the percoll, then use1x1640(with 10% FBS) to make the gradient to isolate immune cells.
And did you get rid of erythrocytes?
Hi Jingjing! We were able to get rid of many of the erythrocytes, but not all. I assume that I will have to optimize the amount of mass I can load per 'column'. I will try using the media to make the gradients.
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