We want to observe dendritic spine morphology on cortical neuron culture. So we transfected cells with plasmids expressing GFP. But, under 63x confocal GFP seems to aggregate into puncta. And the neurite seems to enlarge or swell. Neuron may be not healthy.
We cannot see continuous dendrite. And it's impossible to see spines.
Does it means GFP do something bad to neuron? What can I do to improve my method?
Hi Yang,
Looking at the morphology of your cells they look like neurons gone through necrosis. The puncta labelling you are seing is actually beads, sign of cell death. Do you have control, such as cells from the same batch that did not went through transfection? If you do, do they look healthy or similar? If your controls are healthy then you can check your methods and think about what went wrong or what is killing your cells?
best wishes,
Refik
Hi Yang,
It seems that your cell is dying. Maybe try to purify your DNA and remove endotoxin (If you use Max-prep to amplify your DNA). If this does not work, try another GFP construct.
Best,
Peng
Hi Yang,
As Refik mentioned it is important to know whether not transfected neurons in the same culture look the same or healthier than transfected ones.
Since you need long term cultures to start seeing spines it could be that the media is turning toxic or not supportive for de cells. Other point is when did you transfect the neurons and when do you go to the microscope?
If you nucleofected the neurons at day 0 and checked the cells 15 days later is quite possible that cells will be showing toxic effects from the aggregation of GFP. In general is better when you plate the cells, grow them for 10-15 days an then you transfect the cells by lipofectamine or other similar technics. This method has the drawback of less yield of transfected cells but you won't have issues with toxicity.
It will help if you tell us how you made your cultures (media included), transfection and the timing of the steps in the procedure.
Good luck!
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