Dear Sara,

I dissociate adult tissue for neuronal cell culture, and poor viability can be due to rough dissociation of the tissue or failure to take excitotoxicity into account, which leads to the death of a lot of neurons.  If you search here on RG for tips on culturing neurons, you will find good protocols, as well as suggestions for dissociation methods that preserve viability.  I personally am a fan of the Worthington papain that is tested for CNS dissociation, and the Huettner and Baughman dissociation protocol used for visual cortex.  It can be found in both the first and second editions of the nerve cell culture manual edited by Gary Banker and Kimberly Goslin:

https://mitpress.mit.edu/books/culturing-nerve-cells 

These links may be of help with dissociation technique.:

https://www.researchgate.net/post/Any_advice_on_primary_neuronal_cell_culture_from_embryonic_rat_cortex

Instead of flow cytometry, you may want to investigate the Miltenyi Biotec beads. There may also be other selection protocols that work for your neurons. Links to a few of them can be found at this RG thread:

https://www.researchgate.net/post/What_is_the_best_way_to_enzymatically_dissociate_mouse_brain_tissue_that_gives_a_high_yield_for_all_cell_types_neurons_astrocytes_microglia

As for cell surface markers (your other RG question), this thread may be of help:

https://www.researchgate.net/post/Neuron_surface_marker_antibody_IF_IHC?_tpcectx=profile_answers

I don't know how well the ganglioside survives dissociation protocols, but Thy-1 does since it is used for selection of retinal ganglion calls for culture.

I am sure others here on RG will have helpful information more specific to flow cytometry.

Good Luck!

Jill

Hi Sara,

Our lab works with flow cytometry of brain tissues often. We don't typically sort neurons because, due to concerns of low viability. A few suggestions:

• Intracellular NeuN is probably the best marker, but as the cells cannot be fixed for your downstream applications, this will not solve your specific issue. 
• CD24 may represent a good candidate target, as long as it's combined with others to gate out contaminating glial cells. If you try using CD24+, I would include anti-CD45 (high expression will be peripheral immune cells, intermediate expression would be microglia), anti-CD11b (expressed on MG) and anti-GLAST (surface marker for astrocytes). Then perhaps confirm with NeuN staining on a test panel, to assess neuronal purity. 

Ultimately, the best way may be to perform sorting on transgenic mice that express GFP+ under the control of a neuronal-specific promoter, if you have access to those mice. 

Hi, Sara.

You may adopt our brief procedure to finalize your own protocol.

Pass small chopped pieces of brain gently through 1 ml syringe 3 times, repeat pipettings gently with large-bored blue tip, collect cells, add serum to stop the digestion on ice. Wash the remaining tissues, dissociate them with lower concentration of papain than the suggested with frequent whirling, collect suspended cells, stop the digestion with serum on ice. Repeat the digestion with fresh papain while collecting cells with serum to stop digestion on ice several times. Pull all the cells

Neuronal surface makers, N-Cadeherin & Neurotrimin mixed antibody solution may be used for immunopanning by coating the culture dish to enrich neurons. I have no experience of immunopanning but frequently used in many cell types in the literature. Alternatively, MACS is preferable choice to FACS. You could get enough RNA to do any differential expression studies.

N-Cadherin, you know well. Neurotrimin, mouse gene was cloned and Genbanked by us. It expresses abundantly and also binds to other surface IgSF molecules. If you use 2 antibodies together to enrich neurons, it would make a new protocol established. Try and good luck!