I was wondering if any of you have experience with sorting mature neurons out of a mixed neuron-glia-population by FACS? Which neuronal surface marker would be suitable for such an application? I found some literature where people use CD24 for this. Maybe someone can comment on their own experience?
You could sort the cells based on GFAP expression, thought that isn't a membrane specific protein.
Thre a paper on chick, where they did N-cadherin based MAC sorting of optic tectum neurons. probably you could check if your cells express N-cadherin and use it as a surface marker to sort them.
You can use the MACS (Miltenyi Biotec) system. I am probably going to implement this system soon. They don't sort FOR neurons. If you want Glia, they have antibodies against GLAST that are conjugated to magnetic microbeads . This is used to sort FOR Glia or Oligos, allowing the neurons to flow through the column that is attached to a magnet. Alternatively, if you want the neurons, you can trap the Glia, Oligos, and microglia in the column, and collect the neurons that flow through.
Thank you all for your contribution!
I think I will try the GLAST system, sounds convinient and we have some good experience with the MACS system in other applications. But I will also definitely look for the N-cadherin sorting too!
Thanks again for your help!
the above technique is called Immunopanning. I have a qusetion here that can we isolate brain cells by Percol gradient?
The main problem with FACS separation of mature neurons is the disaggregation of the cells from the culture. Because of the long processes they tend to make clumps.
@Arpita Dave - by percol gradient you can separate only mature oligodendrocytes from the rest of the cells (because of high content of lipids they have different density)
I am also interested by this method and do a UP!
Who tried Miltenyi Biotec material?
I have a follow up question concerning the GLAST system...
Does anybody used the GLAST-MicroBead separation directly on freshly isolated cells (prior to the cell seeding) ?
Hi Casadei,
I am actually isolating neurons since a while, but in the context of a new experimental set-up I would like to have a more or less pure population of neurons and astrocytes from the tissue.
It is interesting!
How do you isolate them usually? Is the method already published by your group?
Still working on this neuron sorting, I found this interesting link for neuron surface merkers!
http://de-de.invitrogen.com/site/de/de/home/References/protocols/neurobiology/neurobiology-protocols/markers-for-characterizing-neural-subtypes.html#prot1
Hi Nils,
How do you isolate your neurons?
By any chance do you know any neuronal membrane marker that I can use to disntiguish axons from dendrites?
Hello to everyone,
was anybody able to isolate adult mouse neurons with FACS?
You can think of selection approach. Synapsin promoter viral vector with puromycin/GFP.
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