Name: isolation of adult human astrocyte populations from fresh-frozen cortex using fluorescence-activated nuclei sorting
Uploaded: 2021-04-16T14:00:00.000+00:00
Duration: 8 min 18 s
Description: Watch this Scientific Journal Video about Isolation of Adult Human Astrocyte Populations from Fresh-Frozen Cortex Using Fluorescence-Activated Nuclei Sorting at JoVE.com

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><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 &amp;micro;M</td><td>SP Scienceware</td><td>136800040</td><td></td></tr><tr><td>DAPI (4&#039;,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&amp;ndash;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 &amp;amp; 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></table>

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

Isolation and Culture of Mouse Cortical Astrocytes

Astrocytes are an abundant cell type in the mammalian brain, yet much remains to be learned about their molecular and functional characteristics. In vitro astrocyte cell culture systems can be used to study the biological functions of these glial cells in detail. This video protocol shows how to obtain pure astrocytes by isolation and culture of mixed cortical cells of mouse pups. The method is based on the absence of viable neurons and the separation of astrocytes, oligodendrocytes and microglia, the three main glial cell populations of the central nervous system, in culture. Representative images during the first days of culture demonstrate the presence of a mixed cell population and indicate the timepoint, when astrocytes become confluent and should be separated from microglia and oligodendrocytes. Moreover, we demonstrate purity and astrocytic morphology of cultured astrocytes using immunocytochemical stainings for well established and newly described astrocyte markers. This culture system can be easily used to obtain pure mouse astrocytes and astrocyte-conditioned medium for studying various aspects of astrocyte biology.

Astrocytes have been recognized to be versatile cells participating in fundamental biological processes that are essential for normal brain development and function, and central nervous system repair. Here we present a rapid procedure to obtain pure mouse astrocyte cultures to study the biology of this major class of central nervous system cells.

Astrocytes  are an abundant cell type in the mammalian brain, yet much remains to be  learned about their molecular and functional characteristics. In ...

Rapid and Refined CD11b Magnetic Isolation of Primary Microglia with Enhanced Purity and Versatility

Microglia are the primary responders to central nervous system insults; however, much remains unknown about their role in regulating neuroinflammation. Microglia are mesodermal cells that function similarly to macrophages in surveying inflammatory stress. The classical (M1-type) and alternative (M2-type) activations of macrophages have also been extended to microglia in an effort to better understand the underlying interplay these phenotypes have in neuroinflammatory conditions such as Parkinson&#39;s, Alzheimer&#39;s, and Huntington&#39;s Diseases. In vitro experimentation utilizing primary microglia offers rapid and reliable results that may be extended to the in vivo environment. Although this is a clear advantage over in vivo experimentation, isolating microglia while achieving adequate yields of optimal purity has been a challenge. Common methods currently in use either suffer from low recovery, low purity, or both. Herein, we demonstrate a refinement of the column-free CD11b magnetic separation method that achieves a high cell recovery and enhanced purity in half the amount of time. We propose this optimized method as a highly useful model of primary microglial isolation for the purposes of studying neuroinflammation and neurodegeneration.

Here, we present a protocol to isolate microglia from postnatal mouse pups (day 1) for in vitro experimentation. This improvised method of isolation generates both high yield and purity, a significant advantage over alternate methods that allows broad range experimentation for the purposes of elucidating microglial biology.

Microglia are the primary responders to central nervous system insults; however, much remains unknown about their role in regulating neuroinflammation. ...

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like mouse and rat astrocyte monocultures: Brain cells can be isolated from different brain regions, and after a week of cell culture, non-astrocytic cells are shaken off by placing the culture dishes on a shaker for 6 h in the incubator. The remaining astrocytes are then passaged into new plates with an astrocyte-specific medium (termed NB+H). NB+H contains low concentrations of heparin-binding EGF-like growth factor (HBEGF), which is used in place of serum in medium. After growing in NB+H, AWESAM astrocytes have a stellate morphology and feature fine processes. Moreover, these astrocytes have more in vivo-like gene expression than astrocytes generated by previously published methods. Ca2+ imaging, vesicle dynamics, and other events close to the membrane can thus be studied in the fine astrocytic processes in vitro, e.g., using live cell confocal or TIRF microscopy. Notably, AWESAM astrocytes also exhibit spontaneous Ca2+ signaling similar to astrocytes in vivo.

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM protocol described here is optimal for culturing murine astrocytes in isolation from other brain cells in a fast, simple, and inexpensive manner. AWESAM astrocytes exhibit spontaneous Ca2+ signaling, morphology, and gene expression profiles similar to astrocytes in vivo.

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like ...

Visualizing and Analyzing Intracellular Transport of Organelles and Other Cargos in Astrocytes

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in brain homeostasis, astrocytes supply neurons with vital metabolites and buffer extracellular water, ions, and glutamate. An integral component of the &#8220;tri-partite&#8221; synapse, astrocytes are also critical in the formation, pruning, maintenance, and modulation of synapses. To enable these highly interactive functions, astrocytes communicate among themselves and with other glial cells, neurons, the brain vasculature, and the extracellular environment through a multitude of specialized membrane proteins that include cell adhesion molecules, aquaporins, ion channels, neurotransmitter transporters, and gap junction molecules. To support this dynamic flux, astrocytes, like neurons, rely on tightly coordinated and efficient intracellular transport. Unlike neurons,&#160;where intracellular trafficking has been extensively delineated, microtubule-based transport in astrocytes has been less studied. Nonetheless, exo- and endocytic trafficking of cell membrane proteins and intracellular organelle transport orchestrates astrocytes&#8217; normal biology, and these processes are often affected in disease or in response to injury. Here we present a straightforward protocol to culture high quality murine astrocytes, to fluorescently label astrocytic proteins and organelles of interest, and to record their intracellular transport dynamics using time-lapse confocal microscopy. We also demonstrate how to extract and quantify relevant transport parameters from the acquired movies using available image analysis software (i.e., ImageJ/FIJI) plugins.

Here we describe an in vitro live-imaging method to visualize intracellular transport of organelles and trafficking of plasma membrane proteins in murine astrocytes. This protocol also presents an image analysis methodology to determine cargo transport itineraries and kinetics.

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in ...

Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive human diseases. Considerable advances have been made to uncover the molecular signatures of homeostatic microglia as well as alterations of their gene expression in response to environmental stimuli. With the advent and maturation of single-cell genomic methodologies, it is increasingly recognized that heterogenous microglia may underlie the diverse roles they play in different developmental and pathological conditions. Further dissection of such heterogeneity can be achieved through efficient isolation of microglia from a given region of interest, followed by sensitive profiling of individual cells. Here, we provide a detailed protocol for the rapid isolation of microglia from different brain regions in a single adult mouse brain hemisphere. We also demonstrate how to use these sorted microglia for plate-based deep single-cell RNA sequencing. We discuss the adaptability of this method to other scenarios and provide guidelines for improving the system to accommodate large-scale studies.

We provide a protocol for isolation of microglia from different dissected regions of an adult mouse brain hemisphere, followed by semi-automated library preparation for deep single-cell RNA sequencing of full-length transcriptomes. This method will help to elucidate functional heterogeneity of microglia in health and disease.

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive ...

Isolation and Direct Neuronal Reprogramming of Mouse Astrocytes

Direct neuronal reprogramming is a powerful approach to generate functional neurons from different starter cell populations without passing through multipotent intermediates. This technique not only holds great promises in the field of disease modeling, as it allows to convert, for example, fibroblasts for patients suffering neurodegenerative diseases into neurons, but also represents a promising alternative for cell-based replacement therapies. In this context, a major scientific breakthrough was the demonstration that differentiated non-neural cells within the central nervous system, such as astrocytes, could be converted into functional neurons in vitro. Since then, in vitro direct reprogramming of astrocytes into neurons has provided substantial insights into the molecular mechanisms underlying forced identity conversion and the hurdles that prevent efficient reprogramming. However, results from in vitro&#160;experiments performed in different labs are difficult to compare due to differences in the methods used to isolate, culture, and reprogram astrocytes. Here, we describe a detailed protocol to reliably isolate and culture astrocytes with high purity from different regions of the central nervous system of mice at postnatal ages via magnetic cell sorting. Furthermore, we provide protocols to reprogram cultured astrocytes into neurons via viral transduction or DNA transfection. This streamlined and standardized protocol can be used to investigate the molecular mechanisms underlying cell identity maintenance, the establishment of a new neuronal identity, as well as the generation of specific neuronal subtypes and their functional properties.

Here we describe a detailed protocol to generate highly enriched cultures of astrocytes derived from different regions of the central nervous system of postnatal mice and their direct conversion into functional neurons by the forced expression of transcription factors.

Direct neuronal reprogramming is a powerful approach to generate functional neurons from different starter cell populations without passing through ...

Efficient Extraction of Astrocytes and Microglia from the Adult Mouse Spinal Cord

Isolation of Pure Astrocytes and Microglia from the Adult Mouse Spinal Cord For In Vitro Assays and Transcriptomic Studies

Astrocytes and microglia play pivotal roles in central nervous system development, injury responses, and neurodegenerative diseases. These highly dynamic cells exhibit rapid responses to environmental changes and display significant heterogeneity in terms of morphology, transcriptional profiles, and functions. While our understanding of the functions of glial cells in health and disease has advanced substantially, there remains a need for in vitro, cell-specific analyses conducted in the context of insults or injuries to comprehensively characterize distinct cell populations. Isolating cells from the adult mouse offers several advantages over cell lines or neonatal animals, as it allows for the analysis of cells under pathological conditions and at specific time points. Furthermore, focusing on spinal cord-specific isolation, excluding brain involvement, enables research into spinal cord pathologies, including experimental autoimmune encephalomyelitis, spinal cord injury, and amyotrophic lateral sclerosis. This protocol presents an efficient method for isolating astrocytes and microglia from the adult mouse spinal cord, facilitating immediate or future analysis with potential applications in functional, molecular, or proteomic downstream studies.

Author Spotlight: Isolation of Glial Cells from the Adult Mouse Spinal Cord 

This protocol outlines the isolation of purified astrocytes and microglia from the adult mouse spinal cord, facilitating subsequent applications such as RNA analysis and cell culture. It includes detailed cell dissociation methods and procedures designed to enhance both the quality and yield of isolated cells.

Astrocytes and microglia play pivotal roles in central nervous system development, injury responses, and neurodegenerative diseases. These highly dynamic ...

Isolation of Microglia from the Cortex of an Adult Mouse Brain

Source: Zhou, L., et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/60347">Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing.</a> J. Vis. Exp. (2019)
The video demonstrates a technique for isolating microglia from the cortex of an adult mouse brain. First, the cortex undergoes segmentation and homogenization to remove neurons and other glial cells, preserving microglia. Following that, the cell suspension is exposed to anti-myelin magnetic microbeads and a magnetic field, effectively separating microglia and myeloid cells from myelin debris and cells containing myelin.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Mechanical Tissue Dissociation
...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Primary Neuronal Culture

A Simple Technique to Isolate and Culture Murine Astrocytes

Source: Wolfes, A. C., et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/56092">Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol.</a> J. Vis. Exp. (2018)
The video demonstrates a simple method to culture murine astrocytes in isolation from other brain cells. During culturing, by using astrocyte-specific media and inhibiting the adherence of non-astrocytic cells, the method selectively cultures astrocytes. These cultured astrocytes exhibit stellate morphology with fine processes.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Rat or Mouse Brain Dissection
...

Astrocyte Isolation: A Method to Obtain Pure Preparation of Mouse Cortical Astrocytes

Source: Schildge, S. et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/50079/isolation-and-culture-of-mouse-cortical-astrocytes">Isolation and Culture of Mouse Cortical Astrocytes.</a> J. Vis. Exp. (2013)
In this video, we describe the method to obtain a pure preparation of astrocytes from newly born mice.

Source: Schildge, S. et al. Isolation and Culture of Mouse Cortical Astrocytes. J. Vis. Exp. (2013)
In this video, we describe the method to obtain a pure ...

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

Summary

Explore More Videos

Chapters in this video

Isolation and Culture of Mouse Cortical Astrocytes

Rapid and Refined CD11b Magnetic Isolation of Primary Microglia with Enhanced Purity and Versatility

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

Visualizing and Analyzing Intracellular Transport of Organelles and Other Cargos in Astrocytes

Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing

Isolation and Direct Neuronal Reprogramming of Mouse Astrocytes

Isolation of Pure Astrocytes and Microglia from the Adult Mouse Spinal Cord For In Vitro Assays and Transcriptomic Studies

Isolation of Microglia from the Cortex of an Adult Mouse Brain

A Simple Technique to Isolate and Culture Murine Astrocytes

Astrocyte Isolation: A Method to Obtain Pure Preparation of Mouse Cortical Astrocytes