Dendritic cell -isolation ---viabillity is very low - Immunology (Oct/20/2004 )
I have prepared splenic dendritic cell using CD11c-magnetic beads & MACS LS coulums.
I can isolate denritic cells using this methods. Purity is higher than 80% checked by Flowcytemetry, viabillity is higher than 95% at this point.
But, this dendritic cells distributed to culture plate, 12 hrs later viabillity becomes lower than 50%. 24hrs later lower than 80%.
Therefore I would like to know how to culture dendritic cells keeps high viabillity. Anyone, please give me tips of dendritic culture methods.
My protocol deteil is below.
Spleen digested by collagenase type 4 37 degrees-15 min
Wash HBSS & lysed red blood cells using ACT lysing buffer.
Wash HBSS & layerd d=1.075 percoll & centrifugation.
Obtained low density cells & CD11c-beads MACS purification.
I suppose you need to keep them alive by stimulating them with cytokines. It depend what you want to to further with them. Maybe if you give them a mixture of gm-csf, il-4 would help them.
i also recommend you reading the paper of romani et. al (pubmed) from 1994, he has used human blood to develop and use dendritic cells.
Hope that this was helpfull to you, if you think you can use this information you can contact me because of the activity and so on of the cytokines.
We have been using cytokines and growth factors from Epoch Biolabs (EGF, FGF, GM-CSF, IL-2, IL-4; epochbiolabs.com) to culture dendritic cells. They are perfectly fine for further in vitro and in vivo experiments.
Thank you for giving advice immediately.
Experiment by carrying out advice to reference.