[Neuroscience] Re: Technical question: dissociated cells

Tom via neur-sci%40net.bio.net (by tehgabriel from web.de)
Fri May 16 03:44:06 EST 2008


On Apr 24, 8:15 am, christinela... from hotmail.com wrote:
> Hi, I'm wondering if anyone here has done electrophysiology on acutely
> dissociated neurons? I've found several methods in the literature and
> have had a couple of attempts with adult rat brain. The cells come out
> looking bright and round and alive, but unfortunately I can't make
> them stick to glass chips, with or without poly-D-lysine coating. I
> need them to stick to something in order to patch them.
>
> Any suggestions?

- Make sure your glas cover slips are really clean (standard protocols
are available in the internet. briefly: sonicate coverslips in a glas
beaker with nitric acid for several hours, rinse with milliq water to
remove the acid, rinse with ethanol, sonicate with ethanol for another
several hours, rinse with fresh ethanol and finally store them in
ethanol. protect cover slips from getting dry at any time point!)

- Try different coating substrates. e.g. d- or l- lysine, or a mix of
collagen and lysine (we use something like 1ml of milliq water with
100 ul rat tail collagen plus 20 ul PDL). in my experience, postnatal
neurons (mouse) don't grow so well on pure PDL coating.

- Check your medium! there are plenty of different receipes how to
prepare culturing medium. In general they should contain some kind of
supplement (N2 or B27) and some serum (horse serum or fetal bovine
serum), diluted in the medium (MEM, neurobasal, etc.)
we use (for 100 ml final volume): 87 ml neurobasal, 10 ml horse serum,
2 ml B27 supplement, and 1 ml glutamax. If you want/need you can add
antibiotics.
This works fine for our postnatal mice hippocampal cultures.

Regards,
Thomas


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