I am going to perform flowcytometry of dendritic cells cultured from PBMC. How can I dissociate the adhered cells? Can we trypsinize? How much percentage trypsin is required? Is there any publication that talks about this?
You should incubate your attached DC in cold PBS at 4ºC around 30' and then washing and harvesting the cells.Adding trypsine would affect some membrane markers for flow cytometry.
Hi,
I recommend to add some EDTA to the PBS. 1 to 2 mM should be sufficient.
The other thing you could consider is growing them up in vitro on PFTE coated plates/culture bags. They're good as plastic can alter the morphology and phenotype of the cell, plus trypsinising can strip off some of the surface molecules. I know you can get the bags from a company called Origen - they are helpful and sent me some testers so you could check it out first!
Cold PBS should be enough for DCs. We are using PFTE culture bags for macrophages which are much more adherent.
Hi Thomas - where are you getting your culture bags from? Our old supplier has stopped making them. A friend in Bristol is going to start making them but so far isn't up and running, any tips on a good supplier? Origen is pretty costly.
Were are using FEP Teflon-coated cell culture bags from CellGenix (number 72-C)
This may help:
http://www.jove.com/video/51554/isolation-human-monocytes-double-gradient-centrifugation-their
Thanks - in my old lab we used to just make them in house so it was dead cheap, but we don't have the kit here so I've been looking for a new good supplier. Origen have amazing stuff but it's a bit overkill for what we need!
I also recommend using EDTA in your PBS,
we use PBS with 2% FCS and 2mM EDTA
with Trypsin you risk shed some surface markers that you might be interested in
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