We had good results with this protocol:

Coat vessels with poly-L-ornithine and laminin:

Add sufficient poly-L-ornithine solution (Sigma-Aldrich P4957) to completely

cover the surface of the wells.

Incubate plates at room temperature for 1 hour.

Dilute laminin stock solution (1 mg/ml, Sigma-Aldrich L2020) to 3.3 μg/ml with

sterile water (1:300 dilution). Aliquot remaining stock solution and store at 20°C.

Remove poly-L-ornithine solution from the wells and wash wells twice with

sterile water.

Add enough diluted laminin solution (3.3 μg/ml) to completely cover the wells.

Incubate plates at 37°C for at least 1 hour or at 4°C over night.

Remove laminin solution from the wells immediately before plating the cells.

Do not allow the surface to dry.

I also heard that matrigel can do the job.

Good luck

Oliver

Have you tried growing fibroblasts on the coverslip, then releasing them using chelating agents or removing them using detergent, leaving behind a matrix that can be washed; the test cells can then be plated upon the prepared 'biomatrix'.

Hi Ira - Thanks. I haven't. Will give it a shot. Very interesting idea and concept.

Appreciate that!

Dear Sray,

we had similar problems. We found that the kex factor ist to use as much medium as possible over the cells for good cell survival

My experience of culturing neurons is on Poly-D-Lysine coated glass cover slips. Poly-L-Lysine may not work. Just to clarify your Poly-Lysine!

We had good results with this protocol:

Coat vessels with poly-L-ornithine and laminin:

Add sufficient poly-L-ornithine solution (Sigma-Aldrich P4957) to completely

cover the surface of the wells.

Incubate plates at room temperature for 1 hour.

Dilute laminin stock solution (1 mg/ml, Sigma-Aldrich L2020) to 3.3 μg/ml with

sterile water (1:300 dilution). Aliquot remaining stock solution and store at 20°C.

Remove poly-L-ornithine solution from the wells and wash wells twice with

sterile water.

Add enough diluted laminin solution (3.3 μg/ml) to completely cover the wells.

Incubate plates at 37°C for at least 1 hour or at 4°C over night.

Remove laminin solution from the wells immediately before plating the cells.

Do not allow the surface to dry.

I also heard that matrigel can do the job.

Good luck

Oliver

Hi Kristen, Muralidharan and Oliver - Thanks for the excellent suggestions. Will try and let you know the outcome, soon.

Cheers

Sravi

1. I would first make sure that the glass has been cleansed of any lipids left over from manufacturing. 

2. Acid etch overnight in HCl or Nitric acid (be careful if going the nitric acid route).

3. Wash ~ 10x with lots of water to remove any acids.

4. Coat with poly-ornithine (in Boric acid) at a concentration several fold higher than what you would do for plastics. * this part is very important. * Glass just needs a little more help in getting things to stick. 

5. After coating with PolyOrn rinse 3x with water and you should be good to go. A brief period in 10% FCS prior to plating cells helps a lot but isn't critical. 

Hi, I also induced neuron from ES cell, from my view, gelatin is OK. try coat your CS with PLO first, then gelatin. or just simply gelatin. it works in my hands

All - Thanks a bunch for the suggestions. I could optimize the conditions for the neurons I work with (Motor neurons). Coating with Ornithine for 2 hrs followed by overnight coating with Laminin at a relatively high conc of 20 ug/ml allowed MN to attach well and remain stable for at least a week. Sometimes I find some neurites - kind of floating around at which time I add additional laminin 1-2 ug/ml made up in cell culture medium which seem to help the neurites re-attach (read that in a published paper elsewhere) .

@Monica Yiyun Jiang - Coating with gelatin did not help. Cells did not attach. 

When you read Gary Bankers book "Culturing Nerve Cells", he mentions there that the glass you use is of utmost importance; he gives company names and glass type to use.

I agree that the type of glass makes a big difference. I use German glass from Electron Microscopy Sciences.

I grow primary neurons with Poly-L-Lysine and laminin, so poly-L does work

I prepare coating with 1:100 concentration of P-orn for 6 hours at 37ºC and then, incubate overnight with laminin (Invitrogen 23017015) at the concentration of 1:400

We can do plasma coating on glass cover slips which gives them a very thin active coating. Cells can strongly bind to the activated glass surface when they are in contact. We can send you some for testing.