Hi Hao Chen,

Clustered/clumped neurons are sign of poor coating. That is why without the coverslips you see less clustering - because the dishes are sold precoated to promote attaching of almost any type of cell (neurons included). You have to coat your coverslips previously, but you should try different coatings to see which one is best for your culture. We use poly-orinithine for hippocampal neurons but laminin+poly-lysine for cortical or just poly-lysine for spinal cord neurons (all empirically tested)

I'm pretty sure you'll find the solution if you try that. Other problems could be density of plating or time of addition of Ara-C to stop glial grow.

Good luck!

Jaime

Do you use  poly-lysin coated cover slips or just plane glass? That might explain why you have difference between empty wells and wells with cover slip. 

Hi Hao Chen,

Clustered/clumped neurons are sign of poor coating. That is why without the coverslips you see less clustering - because the dishes are sold precoated to promote attaching of almost any type of cell (neurons included). You have to coat your coverslips previously, but you should try different coatings to see which one is best for your culture. We use poly-orinithine for hippocampal neurons but laminin+poly-lysine for cortical or just poly-lysine for spinal cord neurons (all empirically tested)

I'm pretty sure you'll find the solution if you try that. Other problems could be density of plating or time of addition of Ara-C to stop glial grow.

Good luck!

Jaime

I agree with Jaime.

This can happen when the surface of the plate is not sufficiently adhesive or, even, when the process of physical disaggregation was not enough.

I agree with the coating being a problem, but from my experience, clumped up neurons are also an indication of an "unhealthy" culture, in the sense that the neuronal density that was seeded was too low. In combination with the coating, I'd recommend increasing the number of neurons you plate. Good luck!

I agree with all of the above - but more from me..

1) Do you have a DNAse step in your isolation protocol ? I experienced 'unheathly' cultures as mentioned by Jeannette if DNAse was omitted

2) Switching from poly-l to poly-d helped my hippocampal cultures 

3) finally - if you use AraC as suggested by Jaime - be careful - neurons don't like to much of it !

Best of luck

Stuart

used fire polhished glass pasteur pipet, for more complete separation, or used plastic pippete and do it a smooth flow between the pipett and culture plate before you plant the cells

We had these kind of clusters in one of our studies and we found that cells within these clusters grow very well, its just that they dont proliferate out. Its got to do with the cells/materials interaction, specifically the surface charges of the material which cells are growing on (eg. a cell-suitable surface charge appears on glass after coating with poly-lysine).

I agree with all above suggestions. Coating your coverslips with Lysine/Laminin will certainly improve the cell proliferation. Primary cells spread very well on Laminin. You observed less clusters otherwise mostly because the wells are usually pre-coated. Also, do a good trituration step before plating the cells, as Jorge suggested.

Thank you all for these kind suggestions! All helped us better understand this issue.

We actually pre-coated the coverslip 24 hours before cell extraction with poly-lysine, but I start to doubt how effective our coating was, as Jaime mentioned.

We also had a DNase step and physical disaggregation step, little clustering was found on the first day.

If coating is not your problem, I believe that neurons may be damaged during dissociation. It always induce neurons aggregation. What enzyme did you use for dissociation? It may the main cause of your problem.

Hi, Chao Hsu-Wen! We used typsin to digest them, and also added DNase. But there seem to be some indigestible materials in it, about which I even raised another question (the link below). I'm pretty sure cells were fully separate after digestion. But how does physical damage induce their clustering?

https://www.researchgate.net/post/What_may_form_sticky_white_indigestible_remnant_after_trypsin_digestion_during_primary_neuron_preparation

Trysin is very strong enzyme for cell dissociation. If your treatment takes long time, it will cause cell damage, especially neurons. I had the same problem before, but I have overcome it by change trypsin to papain. You can try papain, because it is a minor enzyme and harmless during enzyme digestion. Please find the recipe of papain solution in this website.

http://nar.oxfordjournals.org/content/early/2012/06/22/nar.gks598.long

The other things could cause neurons to aggregate is if your coverslip is not coated well. Several things could cause this....

1. dirty coverslip (you may want to try nitric acid wash first but I know some people never do acid wash..... their neurons are fine).

2. bad coating solution. We do reuse poly-L-lysine but you may want to use fresh one until you solve your culture problem. The other thing is that I found it is better to dilute poly-L-lysine stock (we make 10 mg/ml in H2O as the stock solution, keep in -20C) in 0.1M sodium borate solution then just water. If diluted in H2O (there was a time I tried to be lazy and not to keep one more solution in the lab), I found the neurons are fine at first (attached perfectly) but start clustering (~4 days)  and aggregating (7-8 days) in culture.

3. Sad but true, some people just cannot do neuron culture. The health condition of everyone's neuron culture is quiet variable  in the same lab by folowing the same protocol and sharing the same reagents. There is just something, everyone has to figure it out. Good luck