Can I just follow the regular protocol in cell line that use EBSS and stave the neuron for several time points? But I don't know whether this kind of harsh condition suitable for neuron? I have tried several times, but it is hard to observe any change of LC3-II in WB or the puncta in IF. And I also have tried rapamycin (200nM), the results remained the same. Can anybody share some experience of autopahgy induction.
Thanks a lot. I am really desperate.
Hi Mengying,
I don´t work directly with autophagy but for what I know Neurons do increase autophagy when stressed. If I were you at this point I would do several timepoints of starvation and different levels of starvation. Are you blocking autophagy before looking for LC3-II?
Alternatively you can look for other stress inducers that you can use or overexpress LC3-II, Beclin or TFEB.
Hope I could help.
Best
Hi Mengying, try the following: Incubate the neurons in NBS (if necessary adjust the osmolarity (we do it with Manitol)) for 2-3 h, in the presence of bafilomycin. I'm certain you will see upregulation on LC3-II on WB. Hope this was helpful!
Many thanks for the valuable suggestions, Dino and Natalia. Currently, I am trying to block the neuronal autophagy with drugs, like Baf or CQ, and still under testing. Neuron experiment are very unstable. Natalia, can I ask the concentration you used of bafilomycin? I am 100nM, and how long you treat the neuron? Thanks again.
Hi Mengying, try 24h 10nm Baf, and then 2-3h serum-free! Natalia
Serum starvation can induce autophagy within an hour or a couple hours. Alternatively there are many pharmacological agents that can be used to induce - rapamycin is most often used.
HI,John~ What I am really thinking about is that the neuronal autophagy is too quick to detect the change of LC3. Since it has been reported that the basal level of autophagy in brain is highly active, it might be better to block the autophagy with or without the induction to compare the change of LC3. That's what I think~ Thanks your suggestions the same.
Rapamycin induces Autophagy at 200 nM for 3-12 h in primary neuronal cells. To see the Autophagy flux you can use bafilomycin 20 nM for 6 h. I Have recently published one paper in Autophagy and I have used the same concentrations and time points. You have to take extra care of Neurol basal media as well B27. if the media or concentration of B27 will get exhausted you will not get autophagy flux. Neurons are very sensitive so your treatment should be on 18-19th day after isolation.Hopefully you will get autophagic flux through western blot.
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