I am currently incubating the glass coverslips with 0.1mg/mL Poly-d-lysine for 1 hour and then with collagen diluted 1:10 for 2 hours at 37 degrees.
While the neurons stick fine for immuno, they seem to move when we try to clamp them. Does anyone have any other recommendations? Matrigel? Laminin?
Thanks!
We actually combine Poly-lysine and poly-L-Ornithine to get a stronger coating. I have never had problem of neurons detaching during patchclamp. Do you deplete glia in your cultures? Do you use a calcium-free external saline when patching ? Good luck
I prefer polyethyleneimine (PEI) as my substrate for neuronal culture. I used 20-50ug/mL for coverslip coating and the source was PEI Max from Polysciences. This ensures stronger attachment of the neurons, even after many weeks. I often had good results initially with PDL but after 10 days or so the neuronal processes became fasciculated.
Hi Diahann,
I realize your question was asked 5 years ago, but in my experience a double coating of Poly-L-Ornithine (PLO) and Laminin has worked well for primary neural stem cells, as well as for neurons differentiated from primary cells on glass coverslips:
1) Coat coverslip in 0.01% Poly-L-Ornithine for 30 min at room temperature
2) Remove PLO and wash twice with PBS
3) Treat coverslips with Laminin diluted to a concentration of 3-6 ng/ml in PBS and incubate for at least 6 hours (preferably overnight )in an incubator before plating your cells
Hope this is able to help someone. Good luck!
Biren
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