how to culture primary neuron in coated coverslip - how to culture primary neuron in coated coverslip (Mar/12/2006 )
I need culture primary neuron in poly-L-lysine coated coverslips but neuron didnot grow on it. I tried few times but all failed. Who can supply a detailed protocol for it, including primary neuron culture (GD17 rat), coating and culture. Or any tip about it is welcomed also.
Thank you so much.
use special chamber slides.
This type of slides looks like 96wells plate but of course it only have 8 well per slides.
When staining the slides, simply remove the chamber and treat the slides as normal microscopic slides.
I hope this may help.
Do you have some more information on these slides?
Manufacturer, supplier, webpage...?
We see that our hippocampal neurons grow much better on LabTek chambered coverslips that on glass coverslips in a 24-well plate. Unfortunately, these LabTek chambers are not suited for immunostaining, or anyway, you cant fix the cells and store them.
culturing primary neurons on poly-l-lysine coated coverslips is rather easy. i did this often a few years back.
write me an email and i will send to you a protocol.
thomas
thomas.lisse@gsf.de
Hi,
I'm doing this at the moment and I thought it could be useful to ask you a few questions: For how long are you coating them? The PLL doesn't adhere to glass as well as it does to plastic. What happened to me when I didn't let it long enough in the incubator was that the neurons after some days aggregated and died. Furthermore, you can try poly-D-lysine as it is less toxic to the cells.
If you still have problems it could be a good idea to describe in detail what are you doing and we see if you should try other conditions.
Hope this helps...
I'm doing this at the moment and I thought it could be useful to ask you a few questions: For how long are you coating them? The PLL doesn't adhere to glass as well as it does to plastic. What happened to me when I didn't let it long enough in the incubator was that the neurons after some days aggregated and died. Furthermore, you can try poly-D-lysine as it is less toxic to the cells.
If you still have problems it could be a good idea to describe in detail what are you doing and we see if you should try other conditions.
Hope this helps...
I have a related question about primary neuronal culture:
I do corical cultures from rat fetuses. So far, my cells are not consistent. Sometimes they spread out individually (great), but sometimes they clump up as clusters. It looks like the coating problem. I coated with poly-l-lysine hydrobromide (high mol. w. pll with enhanced solubility) at 20 ug/ml (higher conc. appeared to harm cells) overnight and washed with water twice. Some people mentioned laminin coating on top of that, I tried (10 ug/ml in media, 1hr, washed with media, and dried) but had more cell clustering.
Do you have suggestions about a coating technique that is consistent with cell spreading (not clumping)?
Thanks a lot, Min