We like to thank members in Pathology and Neurosurgery at the Icahn School of Medicine at Mount Sinai for help with the procurement of de-identified brain tissue and ISMMS&#39;s Flow Cytometry CORE for expert advice. The study was partially funded by NIH RF1DA048810, R01NS106229, R03NS101581 (to N.M.T.), and R61DA048207 (to S.A.).

NOTE: The Program for the Protection of Human Subjects at the Icahn School of Medicine at Mount Sinai (ISMMS) and its Institutional Review Board (IRB) assures the ethical conduct of research and compliance with federal, state, and institutional regulations. In this study, all postmortem specimens used were de-identified, obtained under appropriate consent through the biorepository, and were exempt from &#34;human research&#34; designation by ISMMS&#39;s IRB (HS#14-01007).&#160;
1. Buffer preparation
NOTE:&#160;If performing downstream RNA sequencing, thoroughly treat all workspaces and tools with RNase Decontamination Solution to prevent mRNA degradation.
<ol>
	<li>Prepare lysis buffer.
	<ol>
		<li>Dissolve the following in distilled H2O up to 50 mL: 5.47 g of sucrose (0.32 M final), 250 &#181;L of 1 M CaCl2&#160;(5 mM final), 150 &#181;L of 1 M Mg(CH3COO)2&#160;(3 mM final), 10 &#181;L of 500 mM ethylenediaminetetraacetic acid (EDTA) (0.1 mM final), 500 &#181;L of 1 M Tris-HCl (pH 8) (10 mM final), 50 &#181;L of Triton X-100 (0.1% final), and 17 &#181;L of 3 M dithiothreitol (DTT, 1 mM final, add fresh) (see the Table of Materials).</li>
	</ol>
	</li>
	<li>Prepare sucrose buffer
	<ol>
		<li>Dissolve the following in distilled H2O up to 50 mL: 30.78 g of sucrose (1.8 M final), 150 &#181;L of 1 M Mg(CH3COO)2 (3 mM final), 500 &#181;L of 1 M Tris-HCl (pH 8) (10 mM final), 17 &#181;L of 3 M DTT (1 mM final, add fresh).</li>
	</ol>
	</li>
</ol>
2. Frozen tissue dissociation into single-nucleus suspension
<ol>
	<li>Add 4 mL of ice-cold lysis buffer to a 7 mL glass tissue douncer (grinder), and keep on ice. Dissect approximately 200-400 mg of fresh-frozen human adult cortex, dounce approximately 50x, and transfer the tissue homogenate to a 12 mL ultracentrifuge polypropylene tube.</li>
	<li>Using a 5 mL pipette, add 6.5 mL of ice-cold sucrose buffer to the bottom of the ultracentrifuge tube, taking care not to disturb the layer between sucrose and the tissue homogenate. 
	&#8203;NOTE: If processing additional samples, carefully balance the tubes by weight by adding additional lysis buffer to the lighter sample. Precise balancing of the ultracentrifuge tubes is essential to prevent damage to the rotor and ensure rotation at the proper speed.</li>
</ol>
3. Ultracentrifugation
<ol>
	<li>Ultracentrifuge the lysate at 24,400 rpm (101,814 &#215; g) for 1 h at 4 &#176;C. Carefully aspirate the supernatant and debris without disturbing the pellet. 
	NOTE: A pellet may not be visible if starting with less than 200 mg of tissue.</li>
	<li>Add 600 &#181;L of phosphate-buffered saline (PBS) (Ca2+, Mg2+ free) to the nuclei pellet and incubate on ice for 10 min before resuspending.</li>
	<li>Pipette up and down 50 times to resuspend the nuclei pellet on ice.</li>
	<li>Use a hemocytometer to visualize the intact nuclei under a microscope and to ensure the concentration of the resuspension is above 105 nuclei/mL before proceeding to the next step (combine 10 &#181;L of the resuspended nuclei with 10 &#181;L of trypan blue).</li>
</ol>
4. Antibody incubation
<ol>
	<li>To immunotag the sample for FANS, add 500 &#181;L of the resuspended&#160;sample pellet, 490 &#181;L of 1x PBS (Ca2+, Mg2+&#160;free), 10 &#181;L of 10% BSA (0.1% final), 1 &#181;L of mouse anti-NeuN antibody conjugated to AF555 (NeuN-AF555, 1:1000 final concentration), and 1 &#181;L of mouse anti-PAX6 antibody conjugated to allophycocyanin (PAX6-APC, 1:1000 final concentration). 
	NOTE: Alternative antibodies conjugated to&#160;other fluorophores may be added (see discussion). Here, mouse anti-OLIG2 conjugated to AF488 (1:1000 concentration) was used with favorable results.</li>
	<li>Perform 4&#8242;,6-diamidino-2-phenylindole (DAPI)-only and single-color controls to set up gating parameters, using a small amount of the sample as necessary.
	<ol>
		<li>To perform AF555 single color control, add 20 &#181;L of the resuspended sample pellet, 970 &#181;L of 1x PBS (Ca2+, Mg2+&#160;free), 10 &#181;L of 10% BSA (0.1% final), and 1 &#181;L of NeuN-AF555 antibody (1:1000 concentration).</li>
		<li>To perform APC single color control, add 20 &#181;L of the resuspended sample pellet, 970 &#181;L of 1X PBS (Ca2+, Mg2+&#160;free), 10 &#181;L of 10% BSA (0.1% final), and 1 &#181;L of PAX6-APC antibody (1:1000 concentration).</li>
		<li>To perform DAPI-only control, add 20 &#181;L of the resuspended sample pellet, 970 &#181;L of 1X PBS (Ca2+, Mg2+&#160;free), and 10 &#181;L of 10% BSA (0.1% final) (DAPI will be added later, see 4.4). 
		NOTE: If more than two antibodies are used, performing fluorescence-minus-one (FMO) control is recommended in addition to the single-color controls.</li>
	</ol>
	</li>
	<li>Incubate the samples and the controls with rotation in the dark for 1 h at 4 &#176;C.</li>
	<li>Add DAPI at 1:1000 to all the samples and controls, and proceed with sorting.</li>
</ol>
5. Fluorescence-activated nuclei sorting (FANS)
NOTE: It is recommended to use an institutional flow cytometry facility with assistance from trained personnel unless already proficient in flow cytometry/sorting techniques.
<ol>
	<li>Gate as shown below.
	<ol>
		<li>First, and for each sample, gate by forward scatter (FSC-A) vs. side scatter (SSC-A) to include particles in the appropriate size range for nuclei, thus excluding red blood cells and debris (Figure 1A). 
		NOTE: A very clean nuclei preparation can distinguish between a smaller debris cluster and a nuclei cluster.</li>
		<li>Next, and for each sample, gate by FSC-A vs. FSC-W (or FSC-A vs. FSC-H) and SSC-A vs. SSC-W (or SSC-A vs. SSC-H) to include only singlet nuclei (Figure 1B).</li>
		<li>Next, and for each sample, gate by DAPI to include intact nuclei singlets (DAPI-high gated population), excluding debris (DAPI-low, on left of gated population) and doublets (on right of gated population) (Figure 1C).</li>
		<li>Set further gating based on fluorescence controls, determined visually as distinct clusters on FACS plots.
		<ol>
			<li>Run DAPI-only control to determine background staining of populations, in the absence of antibody (Figure 1D and Figure 2A).</li>
			<li>Run NeuN-AF555-only control to determine the cutoff for NeuN+ staining in the channel for AF555 (Figure 2B).</li>
			<li>Run PAX6-APC-only control to determine the cutoff for PAX6+ staining in the APC channel (Figure 2C).</li>
			<li>Run additional single-color controls if using more antibodies (Figure 2D,E). 
			NOTE: If using more than two antibodies, an FMO control is recommended to visualize any shifts in populations.</li>
		</ol>
		</li>
		<li>Once all controls have been run, draw the gates for NeuN+ and PAX6+ collections above the established thresholds.
		<ol>
			<li>Gate the NeuN+ population to collect neurons (Figure 1E and Figure 2F).</li>
			<li>Gate the PAX6+ population from the NeuN- population to collect astrocytes (Figure 1E,F and Figure 2F).</li>
			<li>Gate and collect additional glial populations (such as oligodendrocyte progenitor cells, as shown here by OLIG2+) from the NeuN-PAX6- population (Figure 1F). 
			NOTE: In case determining appropriate gating cutoffs is difficult with the flow cytometry software due to indistinct populations, it may be helpful to modify the number of events being visualized (either increasing or reducing the number of events on the FANS plot).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Collect samples appropriately based on the intended downstream analysis.</li>
</ol>
6. Collection of FANS populations for downstream molecular analyses
<ol>
	<li>For bulk RNA sequencing, collect 50,000-500,000 nuclei in PBS (see also section 7.1).
	<ol>
		<li>Add 2 mL of sucrose solution, 50 &#181;L of 1 M CaCl2, and 30 &#181;L of 1 M Mg(CH3COO)2, and fill with PBS up to 10 mL.</li>
		<li>Invert and incubate on ice for 15 min, then centrifuge at 900 &#215; g for 10-15 min at 4 &#176;C.</li>
		<li>Aspirate the supernatant, resuspend in 1 mL of RNA-extracting reagent, vortex, freeze on dry ice, and store at -80 &#176;C.</li>
		<li>Alternatively, collect the samples directly into 200 &#181;L of the RNA-extracting reagent. Add the RNA-extracting reagent up to 1 mL after sorting, maintaining a 1:1 ratio of the reagent to sorted sample. Vortex, freeze on dry ice, and store at -80 &#176;C.</li>
	</ol>
	</li>
	<li>For bulk assay for transposase-accessible chromatin using sequencing (ATAC-seq), collect 50,000-75,000 nuclei in PBS in a microcentrifuge tube coated with 5% bovine serum albumin (BSA). Freeze nuclei on dry ice/-80 &#176;C or immediately use them for ATAC preparation.</li>
	<li>For sn RNA-seq or ATAC-seq, collect nuclei in 0.04% BSA in PBS (see also section 7.2).</li>
</ol>
7. Pre-library preparation tips
<ol>
	<li>Bulk nuclear RNA sequencing library preparation
	<ol>
		<li>After collecting in RNA extraction reagent, perform standard phenol/chloroform RNA extraction by adding phenol/chloroform and precipitating RNA from the upper aqueous layer with ethanol, followed by DNase digestion on tube (15 min).</li>
		<li>Perform RNA cleanup and concentrate in a final volume of 15 &#181;L of water. 
		NOTE: Using this method, representative recovery from 300 mg of adult cortex sample is ~300,000 NeuN+ nuclei (15-20 ng/&#181;L total RNA after cleanup and concentration) and ~250,000 PAX6+ nuclei (10-12 ng/&#181;L total RNA after cleanup and concentration). This representative yield can vary greatly based on sample quality, gating stringency, and RNA recovery.</li>
		<li>Perform quantitative polymerase chain reaction (qPCR) prior to sequencing to confirm enrichment of astrocytes based on high differential expression of canonical astrocyte markers (GFAP, SOX9), 10-formyltetrahydrofolate dehydrogenase (ALDH1L1)) and depletion of neuronal and other cell lineage markers (Figure 1G). 
		NOTE: Collection of the double-negative population (NeuN-PAX6-) allows for a more accurate, multitiered qPCR quality control analysis for the relative enrichment of astrocytes (Figure 2H).</li>
		<li>Generate RNA-seq libraries using a kit recommended for low RNA integrity number values, as expected from postmortem samples.</li>
	</ol>
	</li>
	<li>Single-nucleus sequencing library preparation
	<ol>
		<li>Perform sn sequencing through an institutional sequencing core.</li>
		<li>For nanofluidics-based processing and sequencing, use the recommended concentration of 1 &#215; 106 cells/mL in at least 60 &#181;L of 1x PBS + 0.04% BSA for single-nucleus RNA-sequencing (snRNA-seq) and 3 &#215; 106 cells/mL in at least 20 &#181;L of 1x PBS + 0.04% BSA for single-nucleus Assay for Transposase-Accessible Chromatin using sequencing (snATAC-seq).</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Mitchell, A., Roussos, P., Peter, C., Tsankova, N., Akbarian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+future+of+neuroepigenetics+in+the+human+brain.">The future of neuroepigenetics in the human brain.</a> Progress in Molecular Biology and Translational Science. 128, 199-228 (2014).</li><li>Jiang, Y., Matevossian, A., Huang, H. S., Straubhaar, J., Akbarian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+neuronal+chromatin+from+brain+tissue.">Isolation of neuronal chromatin from brain tissue.</a> BMC Neuroscience. 9, 42 (2008).</li><li>Akbarian, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+PsychENCODE+project.">The PsychENCODE project.</a> Nature Neuroscience. 18 (12), 1707-1712 (2015).</li><li>Matevossian, A., Akbarian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+nuclei+isolation+from+human+postmortem+brain+tissue.">Neuronal nuclei isolation from human postmortem brain tissue.</a> Journal of Visualized Experiments: JoVE. (20), e914 (2008).</li><li>Tome-Garcia, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+type-specific+isolation+and+transcriptomic+profiling+informs+glial+pathology+in+human+temporal+lobe+epilepsy.">Cell type-specific isolation and transcriptomic profiling informs glial pathology in human temporal lobe epilepsy.</a> BioRxiv. , (2020).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+characterization+of+progenitor+and+mature+human+astrocytes+reveals+transcriptional+and+functional+differences+with+mouse.">Purification and characterization of progenitor and mature human astrocytes reveals transcriptional and functional differences with mouse.</a> Neuron. 89 (1), 37-53 (2016).</li><li>Sun, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SOX9+Is+an+astrocyte-specific+nuclear+marker+in+the+adult+brain+outside+the+neurogenic+regions.">SOX9 Is an astrocyte-specific nuclear marker in the adult brain outside the neurogenic regions.</a> Journal of Neuroscience. 37 (17), 4493-4507 (2017).</li><li>Manuel, M. N., Mi, D., Mason, J. O., Price, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+cerebral+cortical+neurogenesis+by+the+Pax6+transcription+factor.">Regulation of cerebral cortical neurogenesis by the Pax6 transcription factor.</a> Frontiers in Cellular Neuroscience. 9, 70 (2015).</li><li>Sakurai, K., Osumi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neurogenesis-controlling+factor,+Pax6,+inhibits+proliferation+and+promotes+maturation+in+murine+astrocytes.">The neurogenesis-controlling factor, Pax6, inhibits proliferation and promotes maturation in murine astrocytes.</a> Journal of Neuroscience. 28 (18), 4604-4612 (2008).</li><li>Zhong, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decoding+the+development+of+the+human+hippocampus.">Decoding the development of the human hippocampus.</a> Nature. 577 (7791), 531-536 (2020).</li><li>Pollen, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+identity+of+human+outer+radial+glia+during+cortical+development.">Molecular identity of human outer radial glia during cortical development.</a> Cell. 163 (1), 55-67 (2015).</li><li>Cvekl, A., Callaerts, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PAX6:+25th+anniversary+and+more+to+learn.">PAX6: 25th anniversary and more to learn.</a> Experimental Eye Research. 156, 10-21 (2017).</li><li>Goc, J., Liu, J. Y., Sisodiya, S. M., Thom, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+spatiotemporal+study+of+gliosis+in+relation+to+depth+electrode+tracks+in+drug-resistant+epilepsy.">A spatiotemporal study of gliosis in relation to depth electrode tracks in drug-resistant epilepsy.</a> European Journal of Neuroscience. 39 (12), 2151-2162 (2014).</li><li>Monoranu, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pH+measurement+as+quality+control+on+human+post+mortem+brain+tissue:+a+study+of+the+BrainNet+Europe+consortium.">pH measurement as quality control on human post mortem brain tissue: a study of the BrainNet Europe consortium.</a> Neuropathology and Applied Neurobiology. 35 (3), 329-337 (2009).</li><li>Ninkovic, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcription+factor+Pax6+regulates+survival+of+dopaminergic+olfactory+bulb+neurons+via+crystallin+&#945;A.">The transcription factor Pax6 regulates survival of dopaminergic olfactory bulb neurons via crystallin &#945;A.</a> Neuron. 68 (4), 682-694 (2010).</li></ol>

The authors have nothing to disclose.

Experimental design following the outlined protocol should be finalized after considering several biological and technical factors. Starting tissue samples are fresh-frozen, without having been fixed, and preferably have a short postmortem collection interval to maximize nuclei recovery. Based on experience, a PMI of up to 24 h allows for adequate nuclei recovery; however, a PMI of 12 h or less is preferable to optimize intact nuclei recovery. Additional factors apart from PMI, including temperature of body storage and p...

The molecular complexity of human astrocytes remains poorly defined in primary tissue, requiring better tools for their isolation and characterization at high resolution, both in health and disease. Separation of intact human neurons and glia from their niche has proven difficult due to limited access of fresh brain tissue samples, the heavily interconnected nature of glial and neuronal processes, and inevitable cellular activation during processing, all of which limit the molecular characterization of these cell types ex vivo1. Fluorescence-activated nuclei sorting (FANS) has emerged as an alternative to live-cell sorting, enabling the dissociation and immunotagging of nuclei populations from frozen tissue. In the past decade, FANS has become widely used for isolating and molecularly characterizing human neuronal (NeuN+) nuclei populations in a variety of brain specimens and anatomical regions1,2,3,4.
However, similar methods for isolating specific glial nuclei subpopulations from human cortex have been limited, leading to a relative lack of sophistication in the understanding of astrocyte complexity in both normal and diseased tissues. To this end, a previously published protocol was adapted for isolating human neuronal populations using FANS4, and a method was validated to enrich for astrocytes (and for oligodendroglial progenitors) using a triple-antibody combination, capturing astrocytes in both resting and reactive conditions5. To specifically enrich for astrocytes in the NeuN- fraction, antibodies were used against one of two transcription factors known to be differentially expressed across astrocyte populations, PAX6 or SRY-box transcription factor 9&#160;(SOX9)6,7. PAX6 is highly expressed during early fetal development within radial glia-like progenitors in germinal zones and contributes toward both neurogenesis and gliogenesis8,9,10,11 as well as to retinal neuronal specification12. In the adult, PAX6 is differentially overexpressed in resting human astrocytes6 and shows protein co-expression with glial fibrillary acidic protein (GFAP) in human epilepsy tissue astrocytes13.
This protocol describes the simultaneous isolation of neocortical neuronal and astrocyte-enriched nuclei populations by FANS. Fresh (unfixed) snap-frozen (i.e., fresh-frozen)&#160;postmortem tissue collected from adult cortex is first mechanically and chemically dissociated. After lysis and ultracentrifugation in a sucrose gradient, the cytoplasmic and extracellular components are discarded while the nuclei are retained. Nuclei are then labeled with fluorescently conjugated nuclear antibodies corresponding to the desired target lineages and sorted using FANS. Following this approach, enrichment of astrocytes is demonstrated in the collected PAX6+NeuN- populations, validated both by a targeted qPCR panel as well as by downstream nuclear RNA sequencing.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10x PBS pH 7.2</td>
			<td>Invitrogen</td>
			<td>70013073</td>
			<td></td>
		</tr>
		<tr>
			<td>ANTI-NEUN ANTIBODY CLONE A60</td>
			<td>Millipore</td>
			<td>MAB377A5MI</td>
			<td>mouse anti-NeuN conjugated to a fluorescent compound AF555 (excitation, 553 nm; emission, 568 nm)</td>
		</tr>
		<tr>
			<td>ANTI-OLIG2 ANTIBODY CLONE 211</td>
			<td>Millipore</td>
			<td>MABN50A4MI</td>
			<td>mouse anti-OLIG2 conjugated to a fluorescent compound AF488 (excitation, 499 nm; emission, 520 nm)</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Fisher</td>
			<td>BP9704-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Bright-Line Counting Chamber</td>
			<td>Hausser Scientific</td>
			<td>3110V</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride Anhydrous</td>
			<td>Fisher</td>
			<td>C614-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Strainers, 40 &#181;M</td>
			<td>SP Scienceware</td>
			<td>136800040</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>DL-Dithiothreitol</td>
			<td>Sigma</td>
			<td>43815-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA Library Kit</td>
			<td>Illumina, Nextera</td>
			<td>FC-121&#8211;1030</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse I</td>
			<td>Worthington</td>
			<td>LS002139</td>
			<td></td>
		</tr>
		<tr>
			<td>Dounce Tissue Grinder</td>
			<td>WHEATON</td>
			<td>357542</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS Sorter</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>BD FACSAria III</td>
		</tr>
		<tr>
			<td>Magnesium Acetate Tetrahydrate</td>
			<td>Fisher</td>
			<td>M13-500</td>
			<td></td>
		</tr>
		<tr>
			<td>PAX6 (PAX6/496) - 100 TESTS</td>
			<td>Novus</td>
			<td>NBP234705J</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA Clean &#38; Concentrator</td>
			<td>Zymo Research</td>
			<td>R1013</td>
			<td></td>
		</tr>
		<tr>
			<td>RNaseZap RNase Decontamination Solution</td>
			<td>Invitrogen</td>
			<td>AM9780</td>
			<td></td>
		</tr>
		<tr>
			<td>SMARTer Stranded Total RNA-Seq Kit Pico Input Mammalian</td>
			<td>Clontech Laboratories</td>
			<td>635005</td>
			<td>Fragmentation time of 2.5 minutes, as recommended for low RIN RNA values.</td>
		</tr>
		<tr>
			<td>Sucrose, crystal certified, ACS, 500 mg</td>
			<td>Fisher</td>
			<td>S5500</td>
			<td></td>
		</tr>
		<tr>
			<td>SW 41 Ti Swinging-Bucket Rotor</td>
			<td>Beckman Coulter</td>
			<td>331362</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl, 1M Solution, pH 8.0, Molecular Biology Grade, Ultrapure</td>
			<td>Thermo Scientific</td>
			<td>J22638AE</td>
			<td></td>
		</tr>
		<tr>
			<td>TritonX-100</td>
			<td>Fisher</td>
			<td>BP151-500</td>
			<td>non-ionic surfactant in lysis buffer</td>
		</tr>
		<tr>
			<td>TRIzol LS Reagent</td>
			<td>Invitrogen</td>
			<td>10296028</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol Reagent</td>
			<td>Invitrogen</td>
			<td>15596026</td>
			<td>reagent for isolation of RNA</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution, 0.4%</td>
			<td>Gibco</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman Coulter Optima XE-100</td>
			<td>A94516</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge tubes PP 9/16 X 3-1/2</td>
			<td>Beckman Coulter</td>
			<td>331372</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure Distilled Water (RNAse-, DNAse-free)</td>
			<td>Invitrogen</td>
			<td>10977023</td>
			<td>referred to as distilled water</td>
		</tr>
		<tr>
			<td>Ultrapure EDTA</td>
			<td>Life Technologies</td>
			<td>15576-028</td>
			<td></td>
		</tr>
	</tbody>
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isolation of adult human astrocyte populations from fresh-frozen cortex using fluorescence-activated nuclei sorting

The complexity of human astrocytes remains poorly defined in primary human tissue, requiring better tools for their isolation and molecular characterization. Fluorescence-activated nuclei sorting (FANS) can be used to successfully isolate and study human neuronal nuclei (NeuN+) populations from frozen archival tissue, thereby avoiding problems associated with handling&#160;fresh tissue. However, efforts to similarly isolate astroglia from the non-neuronal (NeuN-) element are lacking. A recently developed and validated immunotagging strategy uses three transcription factor antibodies to simultaneously isolate enriched neuronal (NeuN+), astrocyte (paired box protein 6 (PAX6)+NeuN-), and oligodendrocyte progenitor (OLIG2+NeuN-) nuclei populations from non-diseased, fresh (unfixed) snap-frozen&#160;postmortem human temporal neocortex tissue.
This technique was shown to be useful for the characterization of cell type-specific transcriptome alterations in primary pathological epilepsy neocortex. Transcriptomic analyses confirmed that PAX6+NeuN- sorted populations are robustly enriched for pan-astrocyte markers and capture astrocytes in both resting and reactive conditions. This paper describes the FANS methodology for the isolation of astrocyte-enriched nuclei populations from fresh-frozen human cortex, including tissue dissociation into single-nucleus (sn) suspension; immunotagging of nuclei with anti-NeuN and anti-PAX6 fluorescently conjugated antibodies; FANS gating strategies and quality control metrics for optimizing sensitivity and specificity during sorting and for confirming astrocyte enrichment; and recommended procurement for downstream transcriptome and chromatin accessibility sequencing at bulk or sn resolution. This protocol is applicable for non-necrotic, fresh-frozen, human cortical specimens with various pathologies and recommended postmortem tissue collection within 24 h.

Nuclei were collected from fresh (unfixed) snap-frozen&#160;temporal neocortex tissue with a postmortem collection time of 12 h. After tissue dissociation into nuclei suspension, samples were incubated with antibodies against NeuN, PAX6, and OLIG2, and sorted according to the gating shown in Figure 1 and Figure 2. Nuclei were collected from NeuN+, PAX6+NeuN-, and OLIG2+NeuN- sorted populations (Figure 1E,F and <stro...

Watch this Scientific Journal Video about Isolation of Adult Human Astrocyte Populations from Fresh-Frozen Cortex Using Fluorescence-Activated Nuclei Sorting at JoVE.com

Isolation of Adult Human Astrocyte Populations from Fresh-Frozen Cortex Using Fluorescence-Activated Nuclei Sorting

We have developed a method that enriches for and isolates human astrocyte populations from fresh-frozen tissue for use in downstream molecular analyses.

The complexity of human astrocytes remains poorly defined in primary human tissue, requiring better tools for their isolation and molecular ...

isolation-adult-human-astrocyte-populations-from-fresh-frozen-cortex

Research

JoVE Journal

Neuroscience

3.3K Views.  Icahn School of Medicine at Mount Sinai. We have developed a method that enriches for and isolates human astrocyte populations from fresh-frozen tissue for use in downstream molecular analyses. 

Video: Isolation of Adult Human Astrocyte Populations from Fresh-Frozen Cortex Using Fluorescence-Activated Nuclei Sorting

An Optimized Procedure for Fluorescence-activated Cell Sorting (FACS) Isolation of Autonomic Neural Progenitors from Visceral Organs of Fetal Mice

During development neural crest (NC)-derived neuronal progenitors migrate away from the neural tube to form autonomic ganglia in visceral organs like the intestine and lower urinary tract. Both during development and in mature tissues these cells are often widely dispersed throughout tissues so that isolation of discrete populations using methods like laser capture micro-dissection is difficult. They can however be directly visualized by expression of fluorescent reporters driven from regulatory regions of neuron-specific genes like Tyrosine hydroxylase (TH). We describe a method optimized for high yields of viable TH+ neuronal progenitors from fetal mouse visceral tissues, including intestine and lower urogenital tract (LUT), based on dissociation and fluorescence-activated cell sorting (FACS). 
The Th gene encodes the rate-limiting enzyme for production of catecholamines. Enteric neuronal progenitors begin to express TH during their migration in the fetal intestine1 and TH is also present in a subset of adult pelvic ganglia neurons2-4 . The first appearance of this lineage and the distribution of these neurons in other aspects of the LUT, and their isolation has not been described. Neuronal progenitors expressing TH can be readily visualized by expression of EGFP in mice carrying the transgene construct Tg(Th-EGFP)DJ76Gsat/Mmnc1. We imaged expression of this transgene in fetal mice to document the distribution of TH+ cells in the developing LUT at 15.5 days post coitus (dpc), designating the morning of plug detection as 0.5 dpc, and observed that a subset of neuronal progenitors in the coalescing pelvic ganglia express EGFP. 
To isolate LUT TH+ neuronal progenitors, we optimized methods that were initially used to purify neural crest stem cells from fetal mouse intestine2-6. Prior efforts to isolate NC-derived populations relied upon digestion with a cocktail of collagenase and trypsin to obtain cell suspensions for flow cytometry. In our hands these methods produced cell suspensions from the LUT with relatively low viability. Given the already low incidence of neuronal progenitors in fetal LUT tissues, we set out to optimize dissociation methods such that cell survival in the final dissociates would be increased. We determined that gentle dissociation in Accumax (Innovative Cell Technologies, Inc), manual filtering, and flow sorting at low pressures allowed us to achieve consistently greater survival (&gt;70% of total cells) with subsequent yields of neuronal progenitors sufficient for downstream analysis. The method we describe can be broadly applied to isolate a variety of neuronal populations from either fetal or adult murine tissues.

An Optimized Procedure for Fluorescence-activated Cell Sorting FACS Isolation of Autonomic Neural Progenitors from Visceral Organs of Fetal Mice

An optimized procedure to purify neural crest-derived neuronal progenitors from fetal mouse tissues is described. This method takes advantage of expression from fluorescent reporter alleles to isolate discrete populations by fluorescence-activated cell sorting (FACS). The technique can be applied to isolate neuronal subpopulations throughout development or from adult tissues.

During development neural crest (NC)-derived neuronal progenitors migrate away from the neural tube to form autonomic ganglia in visceral organs like the ...

Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection

Microdissection is a novel technique that can isolate specific regions of a tissue and eliminate contamination from cellular sources in adjacent areas. This method was first utilized in the study of Nestin-expressing progenitors (NEPs), a newly identified population of cells in the cerebellar external germinal layer (EGL). Using microdissection in combination with fluorescent-activated cell sorting (FACS), a pure population of NEPs was collected separately from conventional granule neuron precursors in the EGL and from other contaminating Nestin-expressing cells in the cerebellum. Without microdissection, functional analyses of NEPs would not have been possible with the current methods available, such as Percoll gradient centrifugation and laser capture microdissection. This technique can also be applied for use with various tissues that contain either recognizable regions or fluorescently-labeled cells. Most importantly, a major advantage of this microdissection technique is that isolated cells are living and can be cultured for further experimentation, which is currently not possible with other described methods.

Nestin-expressing&#160;progenitors are a newly identified population of neuronal progenitors in the developing cerebellum. Using the microdissection technique presented here in combination with fluorescent-activated cell sorting, this cell population can be purified with no contamination from other cerebellar regions and can be cultured for further studies.

Microdissection is a novel technique that can isolate specific regions of a tissue and eliminate contamination from cellular sources in adjacent areas. ...

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.

The goal of this protocol is to use the fluorescence activated cell sorting (FACS) technique to sort specific types of neural cells for subsequent analysis of cell-type-specific gene expression, epigenetic markers, and or protein expression.

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant ...

Fluorescence Activated Cell Sorting (FACS) and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

The study of neuroplasticity and molecular alterations in learned behaviors is switching from the study of whole brain regions to the study of specific sets of sparsely distributed activated neurons called neuronal ensembles that mediate learned associations. Fluorescence Activated Cell Sorting (FACS) has recently been optimized for adult rat brain tissue and allowed isolation of activated neurons using antibodies against the neuronal marker NeuN and Fos protein, a marker of strongly activated neurons. Until now, Fos-expressing neurons and other cell types were isolated from fresh tissue, which entailed long processing days and allowed very limited numbers of brain samples to be assessed after lengthy and complex behavioral procedures. Here we found that yields of Fos-expressing neurons and Fos mRNA from dorsal striatum were similar between freshly dissected tissue and tissue frozen at -80 &#186;C for 3 - 21 days. In addition, we confirmed the phenotype of the NeuN-positive and NeuN-negative sorted cells by assessing gene expression of neuronal (NeuN), astrocytic (GFAP), oligodendrocytic (Oligo2) and microgial (Iba1) markers, which indicates that frozen tissue can also be used for FACS isolation of glial cell types. Overall, it is possible to collect, dissect and freeze brain tissue for multiple FACS sessions. This maximizes the amount of data obtained from valuable animal subjects that have often undergone long and complex behavioral procedures.

Fluorescence Activated Cell Sorting FACS and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

Here we present a Fluorescence Activated Cell Sorting (FACS) protocol to study molecular alterations in Fos-expressing neuronal ensembles from both fresh and frozen brain tissue. The use of frozen tissue allows FACS isolation of many brain areas over multiple sessions to maximize the use of valuable animal subjects.

The study of neuroplasticity and molecular alterations in learned behaviors is switching from the study of whole brain regions to the study of specific ...

Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). We have identified five steps in the workflow that are crucial for obtaining RNA isolates from enteric ganglia with sufficiently high quality and quantity for RNA-seq. First, when preparing intestinal tissue, each sample must have all excess liquid removed by blotting prior to flattening the serosa as much as possible across the bottom of large base molds. Samples are then quickly frozen atop a slurry of dry ice and 2-methylbutane. Second, when sectioning the tissue, it is important to position cryomolds so that intestinal sections parallel the full plane of the myenteric plexus, thereby yielding the greatest surface area of enteric ganglia per slide. Third, during LCM, polyethylene napthalate (PEN)-membrane slides offer the greatest speed and flexibility in outlining the non-uniform shapes of enteric ganglia when collecting enteric ganglia. Fourth, for distinct visualization of enteric ganglia within sections, ethanol-compatible dyes, like Cresyl Violet, offer excellent preservation of RNA integrity relative to aqueous dyes. Finally, for the extraction of RNA from captured ganglia, we observed differences between commercial RNA extraction kits that yielded superior RNA quantity and quality, while eliminating DNA contamination. Optimization of these factors in the current protocol greatly accelerates the workflow and yields enteric ganglia samples with exceptional RNA quality and quantity.

The goal of this protocol is to obtain high-integrity RNA samples from enteric ganglia isolated from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). This protocol involves preparing flash-frozen samples of human intestinal tissue, cryosectioning, ethanolic staining and dehydration, LCM, and RNA extraction.

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue ...

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a powerful tool for studying transcriptional profiles of cells, particularly in heterogeneous tissues such as the central nervous system. However, dissociation methods required for single cell sequencing can lead to experimental changes in the gene expression and cell death. Furthermore, these methods are generally restricted to fresh tissue, thus limiting studies on archival and bio-bank material. Single nucleus RNA sequencing (snRNA-Seq) is an appealing alternative for transcriptional studies, given that it accurately identifies cell types, permits the study of tissue that is frozen or difficult to dissociate, and reduces dissociation-induced transcription. Here, we present a high-throughput protocol for rapid isolation of nuclei for downstream snRNA-Seq. This method enables isolation of nuclei from fresh or frozen spinal cord samples and can be combined with two massively parallel droplet encapsulation platforms.

Here, we present a protocol to rapidly isolate high-quality nuclei from the fresh or frozen tissue for downstream massively parallel RNA sequencing. We include detergent-mechanical and hypotonic-mechanical tissue disruption and cell lysis options, both of which can be used for isolation of nuclei.

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a ...

Primary Cell Culture of Purified GABAergic or Glutamatergic Neurons Established through Fluorescence-activated Cell Sorting

The overall goal of this protocol is to generate purified neuronal cultures derived from either GABAergic or glutamatergic neurons. Purified neurons can be cultured in defined media for 16 days in vitro and are amenable to any analyses typically performed on dissociated cultures, including electrophysiological, morphological and survival analyses. The major advantage of these cultures is that specific cell types can be selectively studied in the absence of complex external influences, such as those arising from glial cells or other neuron types. When planning experiments with purified cells, however, it is important to note that neurons strongly depend on glia-conditioned media for their growth and survival. In addition, glutamatergic neurons further depend on glia-secreted factors for the establishment of synaptic transmission. We therefore also describe a method for co-culturing neurons and glial cells in a non-contact arrangement. Using these methods, we have identified major differences between the development of GABAergic and glutamatergic neuronal networks. Thus, studying cultures of purified neurons has great potential for furthering our understanding of how the nervous system develops and functions. Moreover, purified cultures may be useful for investigating the direct action of pharmacological agents, growth factors or for exploring the consequences of genetic manipulations on specific cell types. As more and more transgenic animals become available, labeling additional specific cell types of interest, we expect that the protocols described here will grow in their applicability and potential.

This protocol describes a cell sorting based method for the purification and culture of fluorescent GABAergic or glutamatergic neurons from the neocortex and hippocampus of postnatal mice or rats.

The overall goal of this protocol is to generate purified neuronal cultures derived from either GABAergic or glutamatergic neurons. Purified neurons can ...

Obtaining Human Microglia from Adult Human Brain Tissue

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining homeostasis, and during infection or injury. Several independent research groups have highlighted the central role that microglia play in autoimmune diseases, autoinflammatory syndromes and cancers. The activation of microglia in some neurological diseases may directly participate in pathogenic processes. Primary microglia are a powerful tool to understand the immune responses in the brain, cell-cell interactions and dysregulated microglia phenotypes in disease. Primary microglia mimic in vivo microglial properties better than immortalized microglial cell lines. Human adult microglia exhibit distinct properties as compared to human fetal and rodent microglia. This protocol provides an efficient method for isolation of primary microglia from adult human brain. Studying these microglia can provide critical insights into cell-cell interactions between microglia and other resident cellular populations in the CNS including, oligodendrocytes, neurons and astrocytes. Additionally, microglia from different human brains may be cultured for characterization of unique immune responses for personalized medicine and a myriad of therapeutic applications.

This protocol is an efficient, cost effective and robust method of isolating primary microglia from live, adult, human brain tissue. Isolated primary human microglia can serve as a tool for studying cellular processes in homeostasis and disease.

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining ...

Fluorescence-Activated Cell Sorting-Radioligand Treated Tissue (FACS-RTT) to Determine the Cellular Origin of Radioactive Signal

Glial cells probably have a considerable implication in the pathophysiology of neurodegenerative disorders, such as Alzheimer's disease (AD). Their alterations are perhaps associated with a pro-inflammatory state. The TgF344-AD rat strain has been designed to express human APP and human PS1&#916;E9 genes, encoding for amyloid proteins A&#946;-40 and A&#946;-42 and displays amyloid pathology and cognitive deficits with aging. The TgF344-AD rat model is used in this study to evaluate the cellular origin of the 18 kDa translocator protein (TSPO, a marker of glial cell activation) binding, and the 5HT2A-receptor (5HT2AR) serotonin receptor levels that are possibly disrupted in AD. The technique presented here is Fluorescence-Activated Cell Sorting to Radioligand Treated Tissue (FACS-RTT), a quantitative cell-type-specific technique complementary to in vivo PET or SPECT or ex vivo/in vitro autoradiography techniques. It quantifies the same radiolabeled tracer used prior for imaging, using a &#947; counter after cytometry cell sorting. This allows determining the cellular origin of the radiolabeled protein with high cellular specificity and sensitivity. For example, studies with FACS-RTT showed that (i) the increase in TSPO binding was associated with microglia in a rat model of lipopolysaccharide (LPS)-induced neuroinflammation, (ii) an increase in TSPO binding at 12- and 18-months was associated with astrocytes first, and then microglia in the TgF344-AD rats compared to wild type (WT) rats, and (iii) the striatal density of 5HT2AR decreases in astrocytes at 18 months in the same rat AD model. Interestingly, this technique can be extended to virtually all radiotracers.

Fluorescence-Activated Cell Sorting-Radioligand Treated Tissue FACS-RTT to Determine the Cellular Origin of Radioactive Signal

Fluorescence-Activated Cell Sorting-Radioligand Treated Tissue (FACS-RTT) is a powerful tool to study the role of the 18 kDa translocator protein or Serotonin 5HT2A-receptor expression in Alzheimer&#39;s Disease at a cellular scale. This protocol describes the ex-vivo application of FACS-RTT in the TgF344-AD rat model.

Glial cells probably have a considerable implication in the pathophysiology of neurodegenerative disorders, such as Alzheimer's disease (AD). Their ...

Gentle Isolation of Nuclei from the Brain Tissue of Adult African Turquoise Killifish, a Naturally Short-Lived Model for Aging Research

Studying brain aging at single-cell resolution in vertebrate systems remains challenging due to cost, time, and technical constraints. Here, we demonstrate a protocol to generate single-nucleus RNA sequencing (snRNA-seq) libraries from the brains of the naturally short-lived vertebrate African turquoise killifish Nothobranchius furzeri. The African turquoise killifish has a lifespan of 4-6 months and can be housed in a cost-effective manner, thus reducing cost and time barriers to study vertebrate brain aging. However, tailored protocols are needed to isolate nuclei of sufficient quality for downstream single-cell experiments from the brain of young and aged fish. Here, we demonstrate an empirically optimized protocol for the isolation of high-quality nuclei from the brain of adult African turquoise killifish, a critical step in the generation of high-quality single nuclei omic libraries. Furthermore, we show that the steps to reduce contaminating background RNA are important to clearly distinguish cell types. In summary, this protocol demonstrates the feasibility of studying brain aging in non-traditional vertebrate model organisms.

Here we present a protocol to isolate nuclei from the brains of the short-lived vertebrate model Nothobranchius furzeri for downstream applications such as single-nucleus RNA sequencing or single-nucleus assay for transposase-accessible chromatin with sequencing (ATAC-seq).