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K. Kangwantas's Content

There have been 3 items by K. Kangwantas (Search limited from 05-April 19)


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#27644 neuronal cultures detached from laminin- and fibronectin-coated plate but not po

Posted by K. Kangwantas on 26 June 2009 - 01:06 AM in Neuroscience

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



#27098 neuronal cultures detached from laminin- and fibronectin-coated plate but not po

Posted by K. Kangwantas on 19 June 2009 - 01:21 AM in Neuroscience

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.



#27005 neuronal cultures detached from laminin- and fibronectin-coated plate but not po

Posted by K. Kangwantas on 18 June 2009 - 02:02 AM in Neuroscience

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 :lol:

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

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




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