Dear Nathanael, once I have supervised a master's thesis which was only about establishing protocols to isolate and prepare for and analyze neuronal cells by flow cytometry.

In the beginning I thought this would be no big deal. There was some literature available suggesting different approaches to prepare single cells suspensions of intact neuronal cells and to get rid of debris. We tried them and established some others on our own. My feeling is, they all kind of work, but none of them was absolutely reliable. In the end you have to be able to trust a method and the results it produces and for us this did not completely seem to be the case for flow cytomety with neurons.

Neurons in adult animals are highly cross linked which makes it complicated to separate them completely without causing severe cell damage.

Most methods employ enzymatic digestions which also by chance digest your epitope of interest. Of course this is true for all single cell suspensions generated out of tissue, but since neurons are already more susceptible to damage and stronger cross linked then many other cell types the choice of a suitable enzymes is already restricted and the chance that none of the epitopes you plan to address will be digested is not to high.

The question is, why you are interested to employ this method. If you can answer this, maybe I can suggest an alternative which does not require to generate a single cell suspension

Hi Nathanael - just sent you an email about this. I think I can help. Cheers, -Kara

In the 70ties, which is long ago, we did it. However with the apparatus of that time we shoot the nucleus out of the cell. So, check the waist bottles!

Nathanael, Check out this recent publication in JNeurochem for some ideas on the technique in CNS tissue (http://www.ncbi.nlm.nih.gov/pubmed/23895375).

Check out this: http://www.ncbi.nlm.nih.gov/pubmed/15664173

Cheers, Christoph

Pick an appropriate antibody for the cells of interest is the most important step. Make sure it's flow cytometry verified.

Dear Nathanael, once I have supervised a master's thesis which was only about establishing protocols to isolate and prepare for and analyze neuronal cells by flow cytometry.

In the beginning I thought this would be no big deal. There was some literature available suggesting different approaches to prepare single cells suspensions of intact neuronal cells and to get rid of debris. We tried them and established some others on our own. My feeling is, they all kind of work, but none of them was absolutely reliable. In the end you have to be able to trust a method and the results it produces and for us this did not completely seem to be the case for flow cytomety with neurons.

Neurons in adult animals are highly cross linked which makes it complicated to separate them completely without causing severe cell damage.

Most methods employ enzymatic digestions which also by chance digest your epitope of interest. Of course this is true for all single cell suspensions generated out of tissue, but since neurons are already more susceptible to damage and stronger cross linked then many other cell types the choice of a suitable enzymes is already restricted and the chance that none of the epitopes you plan to address will be digested is not to high.

The question is, why you are interested to employ this method. If you can answer this, maybe I can suggest an alternative which does not require to generate a single cell suspension

Thanks Ingo for your comments, I suspected that single cell suspension might be troublesome for neurons. I was considering doing multiple samples of different areas of the brain and then trying to characterize the cell population area differences caused by different perinatal treatments. Flow cytometry would allow for characterisation of cell population numbers, rather than just using gene expression to a surrogate marker.

Hi Nathanael:

If you can use nuclear markers try to use the protocol describes here: http://www.ncbi.nlm.nih.gov/pubmed/23616554

Quick and easy even whith adult samples

Hi Nathanael,

Adding to the previous answer, if you're fine using intracellular antigens, which seems to be the case in the paradigm you describe, the following protocol by us enables the very robust detection of a range of intracellular neural antigens:

http://www.ncbi.nlm.nih.gov/pubmed/23826393

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0068519

For instance, MAP2 and GFAP work well and without background when meticulously following the described steps (http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068519.g004/originalimage). In the meantime, we have used it also for a range of nuclear epitopes (e.g., KI67 as a proliferation marker in your setting?).

As you point out, achieving a viable/largely unharmed single cell suspensions of adult cortical neurons may be challenging. I've previously successfully used papain solutions, but that was using olfactory bulb neurons.

A paper comparing different enzymatic treatments would be Panchision et al.:

http://www.ncbi.nlm.nih.gov/pubmed/17332513

We routinely use TrypLE in our (stem cell-based) neuronal cell isolation protocols. Of course, if reverting to intracellular markers, maintenance of surface epitopes may, conveniently, not be relevant.

I'd be very much interested in hearing how this goes. Any other questions let me know any time. Best of luck!!

I do not know how to isolate live neurons, but you can isolate all type of glia for flow cytometry (and they are viable) using homogenization with tripsin and a 75/25/0 percoll gradient. We routinely do this and we are able to get enough viable microglia cells for 4 staining of 10 parameters both intracellular and surface antigens from one mice/rat brain. You can get enough cells from even half a brain. If you want to do PCR or a specific subtype of pure glia cell culture you can further purify your population of interest with magnetic beads. If you are interested in a more detailed protocol you can contact me. Good luck!