DRG neuron adherence issue in culture

Hi all,

We are getting some really great neuronal cultures, but once we begin the fixation and ICC processing, the neurons come off the coverslip.
We are fixing with cold 10% formalin, and using 1X PBS for washes. The ICC protocol is otherwise standard, and the neurons that remain have good staining.
I was reading that adding CaCl (0.1mM) and MgCl (1mM) to the 1X PBS may help with the cell adherence. Does anybody have any success with working with these additives in wash buffers? Or any other tips to improve cell adherence?

Thanks for your help!

Hi Stephanie,
I think the same issue came up a while back on the forum:

The main takeaway, if I’m not mistaken, was that coverslip coating prior to plating was key, and that a fresh batch of Laminin solved the issue at hand.
My own personal anecdata: I follow a similar pre-coating protocol outlined by Alex in that post and I usually don’t have any issues with detachment. I also cut a few corners, ie washing twice instead of three times and using straight PBS instead of PBS-tween for my primary and secondary dilutions. No experience with CaCl or MgGl to add to the conversation unfortunately. Good luck!

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@sshiers Not an uncommon problem.
See this post Primary mouse DRG immunocytochemistry troubleshooting - cell detachment - #5 by achamess
I followed the protocol in the linked paper. PDL, then laminin, no washes of the laminin. Just DRG directly applied after aspirating laminin. Make sure reagents fresh. I used 4% PFA for fixation. Be super gentle with your washes.