Technical question: dissociated cells

[christi...@hotmail.com]

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?

[J.A.Legris]

How about a micropipette under low negative pressure.

--
Joe

[anju...@yahoo.com]

Hi ,
we are using newborn hipppocampal neurons for patching at JCSMR, ANU,
Canberra. they grow well and stick to glass coverslipes as well.
Though I am new student here and learning patching here. I don't have
the references with me rightnow but i can send you them if you wish.

Cheers
Anjum

[Ewa Z-ska]

Hi,
You may want to try approaching your cells with low positive pressure. Test
if they are sensitive to the pressure in your recording pipette. Cells
should stick to the poly-lys coverslips. Give them a few hours to do it so.

best,
eva

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--
Ewa

[Tom]

- 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

[christi...@hotmail.com]

Hey thanks for all the suggestions! I've tried lower/no positive
pressure and still ended up chasing cells around the dish. I shall try
leaving them on the chips for longer and maybe different chip coatings
next.

Thanks again
-C