Our lab has sorted microglia, astrocytes, and neurons from the rat brain using CD11b, GLT-1, and Thy1, respectively, and it works great! See the Schwarz, Smith, & Bilbo (2013) paper below:

http://link.springer.com/article/10.1007/s00213-013-3180-z

Our lab has performed cell sorting for gene expression and ChIP analysis using nuclei isolated from newborn mouse forebrain. For sorting we used NeuN (neurons), GFAP (astrocytes) and Olig1 (oligodendryoctes) antibodies and obtained the expected proportion of each cell type, so it appeared that our method was specific and reliable. We are also using Aldh1l1 as an astrocyte-specific marker, which may also be helpful in sorting astrocytes.

Thats a cool question!

I was also thinking of that. The publications that I found descibing a method for the rat frain is this:

http://www.ncbi.nlm.nih.gov/pubmed/21911005

Additionally there is a recent Neuron paper where they did brain cell FACS analysis:

http://www.sciencedirect.com/science/article/pii/S0896627314001718

Please let me know what works best for your purposes!

Thanks for the responses. If anyone knows a marker for sorting astrocyte nuclei, that would also be very helpful. I did a quick GO analysis of the top astrocyte-specific genes from Ben Barres's microarray/immunopanning/FACS study (Cahoy et al, 2008), and came up with a short list of proteins that should be enriched in astrocyte nuclei, but I haven't tried antibodies against any of them. This would be useful to FAC sort cell-type specific nuclei from human brain for ChIP and other purposes. I hope to try some of them out on adult mouse brain before too long.

Thanks. I've used MACS with streptavidin beads to capture ChTx-biotin-labeled neurons. That works well, and I like Miltenyi's reagents, but I would most like to simultaneously sort multiple cell types.

I am going to try the Miltenyi anti-GLAST antibody conjugated to PE for FACS sorting. I think it's the same one as in the MACS kit. It seems like it should work well, but this will just be from cultured glia; I don't know yet how it might work in brain.

Check out this paper that used Iba1 antibodies to label microglia by immunofluorescence for microglia. They also labeled microglia with fluorescent lectins to perform time-lapse imaging of living tissue.

Article Microglia Regulate the Number of Neural Precursor Cells in t...

Check out this paper that separated neurons, astrocytes, and oligodendrocytes to measure their transcriptomic profiles.

Article A Transcriptome Database for Astrocytes, Neurons, and Oligod...

For neurons NeuN is the most common marker, but in some cases NeuN positivity can disappear even when neurons are still viable:

http://www.ncbi.nlm.nih.gov/pubmed/15223381

To mark astrocytic bodies, we prefer to use S100ß rather than GFAP, which marks fibers:

https://www.researchgate.net/publication/10850264_Visual_deprivation_effects_on_the_s100beta_positive_astrocytic_population_in_the_developing_rat_visual_cortex_a_quantitative_study?ev=prf_pub

https://www.researchgate.net/publication/26270240_Physical_exercise_is_required_for_environmental_enrichment_to_offset_the_quantitative_effects_of_dark-rearing_on_the_S-100beta_astrocytic_density_in_the_rat_visual_cortex?ev=prf_pub

https://www.researchgate.net/publication/257600300_Increased_physical_activity_is_not_enough_to_recover_astrocytic_population_from_dark-rearing._Synergy_with_multisensory_enrichment_is_required?ev=prf_pub

For microglia, Tomato Lectin (LEA) is also widely used, but it also marks endothelial cells, so maybe it is not the best option.

Article Visual deprivation effects on the s100β positive astrocytic ...

Article Physical exercise is required for environmental enrichment t...

Article Increased physical activity is not enough to recover astrocy...

Our lab has sorted microglia, astrocytes, and neurons from the rat brain using CD11b, GLT-1, and Thy1, respectively, and it works great! See the Schwarz, Smith, & Bilbo (2013) paper below:

http://link.springer.com/article/10.1007/s00213-013-3180-z

We were happy with FACS sorting using double stain (IB4 andCD11b) for microglia, GFAP for astrocytes and NeuN for neurons. Selecting culture medium before FACS sorting also helps, depending on which cell type you want to isolate ( if you are interested in one type of cells, which was microglia in our case (see: Moussaud et al. 2010 http://www.ncbi.nlm.nih.gov/pubmed/20097228)

Great, thanks! So far I've seen that the anti-GLAST and anti-CD11B from Miltenyi work great to separate cultured astrocytes from microglia using FACS (APC and PE). I'm waiting on some mouse neurons that I will throw into the mix to test ChTx-488 labeling before trying whole mouse brain using the protocol from Guez-Barber et al (2012) FACS purification of immunolabeled cell types from adult rat brain, J Neurosci Methods.

To sort cells you ideally need a cell-specific, cell surface marker that can be combined with antibodies to pull the cells out from a cell suspension. I would say that the paper of Ben Emery (listed above in Johnathon's comments, and below) outlines this exact problem nicely. For astrocytes Itgb5 is a great marker.

To keep in mind is whether or not you would like to grow your cells after your have purified them, or simply collect RNA in an acute experiment. The reason I ask is that some cell-sorting systems contain conjugates to the antibodies of interest that can cause the cells to die or become reactive - for eg. magnetic beads can play havoc with astrocytes long term.

Article A Transcriptome Database for Astrocytes, Neurons, and Oligod...

There is an interesting new method that can sort cells based on RNA expression. They didn't pay me to advertise this. I just think it looks like a cool new idea.

Thanks! I found that the Miltenyi anti-GLAST and anti-CD11b worked well to separate astrocytes and microglia, respectively, from P2 mouse cortex via FACS. ChTx-488 seemed to also label a unique population (hopefully neurons), while there was some less intense labeling of astrocytes with ChTx, which is consistent with what I've seen in mixed cultures. I captured the cells into Trizol and have yet to do RT-qPCR with cell-type markers (GFAP, NeuN, Iba1 etc). I will try the anti-Itgb5 as well, and I am going to test the anti-CD171 (neuronal) and anti-ASCA2 (astrocytes) antibodies from Miltenyi as well as an anti-CD45 (microglia) from Abcam.

My next step is to separate these populations from adult mouse brain, cortex and hippocampus. Does anyone have protocols for single cell suspensions, mixed populations, from adult rodent brain? This is what I've used so far (Accutase + trituration + Percoll gradient): (Guez-Barber 2012 FACS purification of immunolabeled cell types from adult rat brain J Neurosci Methods). It worked well for neonatal mouse forebrain, but I haven't tried it yet on the adult.

Hi Alison Where your experiments to separate brain cell populations from adult mouse brain successful? I am also interested in this method and hope to get some tips. Thanks, Oded

The GLAST and Cd11b from Miltenyi work really well. For neurons, maybe CD171, but we haven't tested it thoroughly. We're gravitated towards sorting just the nuclei, for which NeuN works very well. There's some work showing that mRNA is obtainable from cells that are permeabilized and labeled with a nuclear marker before FACS (Hrvatin, 2014, PNAS), and we're trying that as an alternative to cell-surface labeling.

Hi Alison.  I wondered whether you had made progress on sorting cells from human brain (or whether you're aware of anyone else working on that)?  Thanks!  -Janna

Hi all!

I and my colleagues are trying to do full transcriptome database for rat astrocytes (isolated by Magnetic-activated cell sorting with ACSA-1(GLAST) from Miltenyi Biotec) from P3 and P11 animals, but we had obtained some strange results, because we detected expression of neuron-specific markers, such as Tubb3, Map-2, Eno2, Dcx.

Please suggest what is the method would be preferable to astrocytes isolation and what level of contamination can gives Magnetic-activated cell sorting?

Hi, Alison. I also want to separate nueron cells from adult mous brain. I wonder whether your protocol works well ? if so, can you give any detailed infromation? Thanks very much.

Hi Alison,

I also want to isolate adult mouse microglia and astrocyte from the same prep for FACs sorting. Did you try GLAST1 and ITGB5 on adult mouse astrocyte isolation? What kind enzyme do you use to dissociate mouse brain to get single cell suspension and the Ab still works to label astrocytes? Thank you so much!