neuronal cultures detached from laminin- and fibronectin-coated plate but not po
Posted 18 June 2009 - 02:02 AM
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.
Posted 18 June 2009 - 06:46 AM
then heaven will be yours, before you meet your end
Posted 18 June 2009 - 09:53 AM
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 [Culturing hippocampal neurons; Kaech and Banker 2006]
Edited by Pyaria, 18 June 2009 - 09:54 AM.
Posted 19 June 2009 - 01:21 AM
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.
Posted 26 June 2009 - 01:06 AM
Posted 22 March 2013 - 10:58 PM
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??