My end goal is to isolate glia and infiltrating lymphocytes from the CNS of my disease model for FACS analysis and adoptive transfer. My hope is to find a method that will keep cells alive through the dissociation process and further positive section using the Milltenyi nanobead column separation technique. Currently I have a protocol that calls for Collagenase D mediated dissociation of CNS tissue followed by density gradient centrifugation. Unfortunately the protocol fails to provide specifics such as Collagenase D and DNase concentrations or centrifuge g force required. Any information is greatly appreciated.
Cheers,
Brian
I had good experience with dissociation kits from Miltenyi. They usually require only minor optimization, and the dissociation is very good, especially for neonatal brains. For adult brains is a bit more tricky and the purity of subsequent enrichment on magnetic beads might be compromised.
Unfortunately I'm unable to comment on the Collagenase D dissociation, as I never used it. I think the G force should be adjusted to the density of your gradient.
Good luck!
Lech
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