Hi everyone,
I would like to hear your opinions and experiences about dissoctiaton and enzymatic digestion of murine neontal (~P2-P8) brain tissue.Our goal is to obtain cell suspensions for flow cytometry with as many as possbile preserved surface markers. We established a neontal CNS infection model and are interested in the immune cell compostion in the CNS upon infection. So far we dissociated brains with a gentleMACS dissociator and digested the tissue with the Neural Tissue Dissociation Kit P, containg papain (Milteny). Unfortunately of all markers we stained for, only CD45.2, CD11b, CX3CR1 and Ly6C were preserved. We know that Ly6G, CD3, CD4, CD8, CCR2, NK1.1 and CD11c have been digested. So papain is absolutely off the table for us.
Any suggestion concerning initial dissociation and subsequent enzymatic digestion is very welcome, as we need to establish a completely new protocoll.
Thanks for your help and best,
Dennis
Hi Dennis,
you can use pluriStrainer (different sizes) to mesh you brain homogenize through. For the digestion you can use Liberase (2U/ml) and for the washing of the cells you should use a buffer containing DNase. That should help. Good luck
Michael
Hey Michael,
thanks for your suggestion! Which Liberase hav you used so far? There are 5 different variations available in the moment and Roche/Sigma officially don't recommend any of them for digestion of neonatal brain tissue.
Best,
Dennis
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