neuronal cultures detached from laminin- and fibronectin-coated plate but not po - (Jun/18/2009 )

I am working with mouse primary cortical neuronal culture.
The problem is after I seed cells onto laminin or fibronectin-coated plates and leave them overnight in the incubator, cells are clumpy the following day.. obviously they don't attach to the plates but they tend to form clusters. On the fifth day, most clusters attach to the plates and elongate processes. This can be seen in laminin and fibronectin-coated plates , but not neuronal cultures in polylysine-coated ones.

Recently I have found this article 'Phosphothioated Oligodeoxynucleotides Induce Nonspecific Effects on Neuronal Cell Adhesion in a Growth Substrate-Dependent Manner'
Briefly, they added CpG (oligonucleotides, TLR9 ligand) to neuronal culture in Polylysine or laminin-coated plates and cell detachment happened in laminin-coated but no Polylysine-coated plates. The photos in this article look exactly like my cultures so I make me think that there may be bacterial DNA (esp. E.coli DNA which is also TLR9 ligand) contamination in something that I have added to cells.

I am thinking also about my plate-coating method may be wrong. I dilute laminin and fibronectin in PBS with Ca++ and Mg++ and add onto the plates, incubate 4'c overnight, block with BSA and wash with PBS without CA and Mg. I have tried coating laminin on top of polylysine but I doesn't solve the problem

Any suggestions of what's happening to my cultures? or source of bacterial DNA contamination?

ps. I am using Neurobasal, B27, PDS, penicillin/streptomycin and glutamine medium// trypsin, DNase and FCS for cell dissociation.

To rule out contamination, you could set up a well of complete media without antibiotics or cells and see if anything grows out. I'm curious why you're adding BSA after coating the plates. I've never seen that before, and I suspect it may be part of the problem. I coat with a .01% Poly-L-Lysine solution and .04mg/mL laminin, let dry overnight, than rinse with DI water. This seems to work well with the DRG neurons I work with. Also, are you using Neurobasal medium or Neurobasal-A? if you work with adult cells, I'd recommend Neurobasal-A, the osmolarity is a little different.

your cells aren't contaminated.

If your cells clump together and form straight neurites a few days after seeding, it's a coating problem (ie, laminin/polylysine)

If, however, they look normal then they start to clump and form straight neurites, they're not healthy with what you've done to them.

When neurons are contaminated.... they just die. >___<

There's a great protocol paper that describes the procedure for culturing rat hippocampal neurons on glass coverslips over a glia feeder layer, but a lot of the steps are also applicable to mouse cortical neuron culture

What I've heard is BSA is added to block the plate..
kinda fill the empty gaps that haven't occupied by laminin or fibronectin.
It is the same as when we add BSA to ELISA plate after coating it with capture antibodies.

I'll try coating plate without BSA. I will post the result here next week.
Thank you very much for good suggestions.

I've tried again yesterday. Without BSA, I still can see floating clusters today.

I'm starting to work with a primary neuronal cultures from DRG cultures. First time I cultured, the cells did not attach at all and died in two days. But then on searching I found coating with laminin/ fibronectin necessary.
I wanted to know if fibronectin works the same as laminin in coating for these primary neuronal cultures. If yes, then what concentration is recommended??