The authors would like to thank Catherine Alford for suggestions on cell dissociation methods and Kevin Weller, David Flaherty and Brittany Matlock for support in the Flow Cytometry Shared Resource at Vanderbilt University Medical Center and Melissa A. Musser for artistic assistance with illustrations. We thank Drs. Jack Mosher and Sean Morrison for advice on implementing isolation of neuronal progenitors. The VMC Flow Cytometry Shared Resource is supported by the Vanderbilt Ingram Cancer Center (P30 CA68485) and the Vanderbilt Digestive Disease Research Center (P30 DK058404). This work was supported by funding from US National Institutes of Health grants DK064251, DK086594, and DK070219.

1. Preparation of Media (All steps done in tissue culture hood)
<ol>
 <li>Combine the following: 44 ml L-15 Medium, 0.5 ml 100X Penicillin/Streptomycin (P/S), 0.5 ml 100 mg/ml Bovine Serum Albumin (BSA), 0.5 ml 1M HEPES, 5 ml Tissue Culture Grade Water. Be sure to mix BSA and P/S thoroughly before adding. This volume should be adequate for dissociation of up to five different tissues types. This media will be used in Step 3.4 to prepare Quenching solutions for use after enzymatic dissociation of tissue.</li>
 <li>Filter Media though 0.22 &mu;m Polyethersulfone (PES) filter.</li>
 <li>Prepare 1X Hank's Balanced Salt Solution (HBSS) and 1X Phosphate Buffered Saline (PBS) from 10X Stocks using tissue culture grade water. Filter through 0.22 &mu;m PES filter. Large volumes of these reagents can be prepared ahead of time and stored at 4 &deg;C, aliquoting smaller volumes in a tissue culture hood as needed.</li>
 <li>Fill multiple 15 ml conical tubes with 1X HBSS, (number of tubes to equal the number of tissues you plan on sub-dissecting) keeping tubes on ice.</li>
</ol>
2. Dissection
<ol>
 <li>Euthanize timed pregnant mouse in accordance with institutional animal care and use committee approved protocols and transfer uterus into a 60 or 100 mm Petri dish containing ice cold 1X PBS.</li>
 <li>Remove embryos from uterus and euthanize by decapitation in ice-cold 1X PBS. Screen individually under fluorescence illumination, dividing into positive transgenic and wild-type (WT) nontransgenic pools. Keep embryos in ice-cold 1X PBS throughout dissection.</li>
 <li>Under a dissecting microscope, sub-dissect the urogenital tract. Hold the embryo in place at the forelimb level with fine forceps. Remove the viscera from the liver down to the genital tubercle by inserting forceps at the level of the diaphragm then briskly pulling the internal organs down and away from the dorsal body wall.</li>
 <li>Further sub-dissect the tissue of interest away from the surrounding tissue (Figure 1). Place each individually sub-dissected tissue type into a 15 ml conical tube containing ice cold 1X HBSS. Pool each tissue type together according to embryo phenotype (i.e. all GFP+ samples of fetal intestine are pooled in a single 15 ml conical tube).</li>
 <li>In parallel, dissect comparable tissues from wild-type embryos to use for compensation controls at flow cytometry.</li>
</ol>
3. Dissociation of Subdissected Tissues
<ol>
 <li>Pellet sub-dissected tissue by centrifugation at 210 relative centrifugal force (rcf), 4 &deg;C for 5 min. Following centrifugation, aspirate off as much HBSS as possible.</li>
 <li>Resuspend the tissue pellet in Accumax (Innovative Cell Technologies, Inc), being sure to change pipette tips between each sample to avoid cross-contamination of tissue types. The amount of Accumax added can be scaled for larger or smaller amounts of tissue. Typically for 1-5 fetal intestine samples, 1 ml of Accumax would be used but larger tissue pools will require a greater volume to achieve dissociation.</li>
 <li>Place tubes into 37 &deg;C water bath for 20-45 min depending on stage of the tissue being isolated (e.g. 13.5 dpc intestine 20 min, 15.5 dpc intestine 35 min). Halfway though the dissociation time, manually break up the tissue by knocking the tube against the side of the water bath (or any solid surface) and by "flicking" the tube (as one would shake down an old -fashioned mercury thermometer). At the end of the dissociation time repeat the shake down procedure multiple times to further dissociate the sample. For more fragile samples, reduce the vigorousness of the shake down and instead use pipette trituration during Step 3.5 to achieve an appropriate level of dissociation. Typical dissociation times for 14.5 dpc and 15.5 dpc LUT are 35 and 45 min, respectively.</li>
 <li>While tissue is incubating in Accumax, make up Quenching solutions, termed Quench and Quench 1:5. Quench is made by adding 45 &mu;l 5 mg/ml DNase I to 6 ml L-15 Media. Quench 1:5 is made by adding 45 &mu;l 5 mg/ml DNase I to 30 ml L-15 Media.</li>
 <li>At end of dissociation, move 15 ml tubes onto ice and immediately add 1 ml Quench to each tube. Triturate each sample up and down until tissue is almost completely dissociated (Figure 2). There will still be some small chunks of tissue present in the solution. Achieving a completely homogenous sample is neither easily achievable nor desirable as it can result in poor cell viability.</li>
 <li>Keep sample on ice as much as possible for the remainder of the protocol. Be sure to use a fresh pipette tip when pipetting up Quench or Quench 1:5 to avoid cross-contamination of samples.</li>
</ol>
4. Filtering Cell Suspension
<ol>
 <li>Using forceps that have been soaked in 70% ethanol, place a 3 cm square of 38 &mu;m nylon mesh membrane (Sefar America) over the mouth of a new 15 ml conical tube. Filter cells through mesh by pipetting into center of membrane using narrow bore tips. If the membrane becomes saturated while filtering, remove it, dry the mouth of the tube with a kimwipe, and use a new piece of nylon mesh to filter the remainder of cell suspension. Once all cells have been filtered, use 1 ml Quench 1:5 to rinse out the tube and filter any remaining cells.</li>
 <li>When filtering is complete, or when replacing a clogged membrane, use forceps to roll up the sides of the mesh and wipe the membrane across top of tube to remove any hanging droplets of cells.</li>
 <li>Pellet cell suspension by centrifugation at 210 rcf, 4 &deg;C for 5 min. Aspirate off supernatant and resuspend pellet in 1 ml Quench 1:5.</li>
 <li>Filter cell suspension through nylon mesh into 5 ml polystyrene tubes. Wash out 15 ml tube with 1 ml Quench 1:5 and filter to capture any remaining cells.</li>
</ol>
5. Preparing Samples for FACS
<ol>
 <li>If using the same tissue for sorting and for compensation controls, transfer 1/10th to 1/20th of the sample volume to a new 5 ml polystyrene tube to use for compensation controls. Divide the wild-type tissue sample into two portions. One portion will be unstained wild-type (WT Only) while the second half will have 7-Aminoactinomycin D (7-AAD, a fluorescent intercalator excluded from live cells used as a "viability stain") added to it (7-AAD only). These individual controls are needed to allow for compensation of any spectral overlap between emission of discrete fluorophores at the flow sorter. For instance, for a single fluorescent reporter like EGFP, necessary controls would include: EGFP only, unstained WT cells, and a tube stained with 7-AAD only (Figure 3).</li>
 <li>Fill all 5 ml tubes containing cell suspension with Quench 1:5 and centrifuge at 210 rcf, 4 &deg;C for 5 min. Aspirate supernatant, leaving approximately 200 &mu;l of liquid in tube.</li>
 <li>Dilute 7-AAD 1:1000 in Quench 1:5. Add 7-AAD staining dilution to 7-AAD only compensation control and samples to be sorted. Do not add 7-AAD to the EGFP only or WT only compensation controls. Volume of 7-AAD to be added will vary based on amount of starting material and size of cell pellet obtained. Once 7-AAD has been added to appropriate tubes, samples are ready to be sorted.
 <table border="1">
 <tbody>
 <tr>
 <td>Tissue</td>
 <td>Sample Pool Size</td>
 <td>Volume 7-AAD to be added to tube*</td>
 </tr>
 <tr>
 <td>15.5 dpc Intestine</td>
 <td>1-5</td>
 <td>200 &mu;l</td>
 </tr>
 <tr>
 <td>15.5 dpc LUT</td>
 <td>1-5</td>
 <td>150 &mu;l</td>
 </tr>
 </tbody>
 </table>
 
 Table 1. Amount of 7-AAD added to different tissue dissociates * The final total amount in each tube will vary by an amount ranging from 50-100 &mu;l since the aspiration of media is an approximate process and performed to avoid the pellet. Final volumes are not measured so as to minimize handling of cells in solution.</li>
 <li>Prepare collection tubes to capture cells for RNA isolation by adding 0.75 ml TRIzol-LS to 1.5 ml microcentrifuge tubes.</li>
 <li>If collecting cells for in vitro culture, rather than RNA isolation, sort cells directly into self-renewal media in 6 well tissue culture plates, coated with fibronectin and filled with media as previously described.2,6,7</li>
</ol>
6. Flow Cytometry
<ol>
 <li>At the flow cytometer, evaluate the compensation controls first, being sure to back flush between each sample to avoid contamination. Use the profiles of the compensation samples to set voltages/ gates for sorting. Note that if positive cells in the tissue desired are present in limiting numbers, a distinct tissue can be used to establish compensation settings as long as the fluorescence intensity and cell size between samples are comparable.</li>
 <li>Set compensation parameters and gates to avoid dead cells that take up 7-AAD dye and collect cells exhibiting high intensity of GFP fluorescence relative to unstained controls (Figure 4).</li>
 <li>Sort maximally 25,000 cells into each microcentrifuge tube. Sorting should be performed at the lowest possible pressure with a wide bore nozzle and low flow rate (e.g., 17psi, 100 &mu;m nozzle, 3,000 events/sec) to preserve neuronal progenitor viability. For sorts to isolate EGFP+ cells we perform our isolations on a BD Biosciences FACSAriaII using a 488nm 20mW laser to excite the EGFP reporter.</li>
 <li>If samples contain high cell concentrations, it is advisable to dilute the cell suspension further using 1:1000 7-AAD stain to achieve higher capture efficiencies when sorting.</li>
 <li>Vortex each tube of cells captured in TRIzol-LS immediately after sorting.</li>
</ol>
7. Representative Results
Tissue dissociation to produce a cell suspensions for flow sorting is a delicate balance between adequate enzymatic digestion and avoiding over-digestion that can result in low cell viabilities. An example of desired level of tissue dissociation is shown in Figure 2. In appropriately digested tissue before manual trituration pieces of sub-dissected organs are still clearly evident (Figure 2b, 2f). In tissues that are enzymatically treated for too long a period of time or at too high a concentration of enzyme, the resulting suspension lacks any residual large pieces of tissue (Figure 2d, 2h).
Appropriate dissociation and manual filtering produce sort profiles at flow cytometry that typically exhibit greater than 90% viable cells and show high levels of EGFP expression (Figure 4). Cell populations obtained by this method illustrate good viability and can be captured for subsequent culture or analysis of gene expression by gating for capture of EGFP+ neuronal progenitors.
<img alt="Figure 1" src="/files/ftp_upload/4188/4188fig1.jpg" /> 
Figure 1. Distribution of TH-EGFP+ neuronal progenitors in fetal mouse LUT. Whole-mount urogenital tract at 15.5 dpc viewed ventrally under bright field illumination (a) compared to distribution of EGFP+ cells labeled by TH-EGFP transgene expression identified under fluorescence illumination (b). TH-EGFP expression is present in adrenals (a) and medially located celiac ganglia (cg). Lateral (c) view of 15.5 dpc TH-EGFP sub-dissected bladder exhibits fluorescence from transgene expression in pelvic ganglia (pg), the bladder wall (bla) and urethra (u). In dorsal view (d) EGFP+ cells are evident in the anterior dorsal urethra. Other labels: kidneys (k), testis (t), bladder (bla) and genital tubercle (gt).
<img alt="Figure 2" src="/files/ftp_upload/4188/4188fig2.jpg" /> 
Figure 2. Brightfield images of 15 dpc fetal LUT (a) and intestine (e), respectively, imaged halfway through the dissociation incubation period, at the end of the dissociation incubation before disruption (b, f), after manual disruption (c, g), and in a sample that has been overly dissociated (d, h).
<img alt="Figure 3" src="/files/ftp_upload/4188/4188fig3.jpg" /> 
Figure 3. Schematic Diagram illustrates compensation controls needed to establish rigorous FACS gating parameters.
<img alt="Figure 4" src="/files/ftp_upload/4188/4188fig4.jpg" /> 
Figure 4. Representative image of flow sort profiles at 14.5 dpc (a) and 15.5 dpc (b). Black population is comprised of single cells based on forward and side scatter that are dead and labeled by 7-AAD fluorescence. Gray population is comprised of singlet cells based on forward and side scatter that have excluded 7-AAD and are thus viable. Green gated population is indicated by boxed "GFP+" area and is comprised of single cells that have excluded 7-AAD (viable) and exhibit EGFP fluorescence.

<ol>
	<li>Gong, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gene+expression+atlas+of+the+central+nervous+system+based+on+bacterial+artificial+chromosomes.">A gene expression atlas of the central nervous system based on bacterial artificial chromosomes.</a> Nature. 425, 917-925 (2003).</li><li>Morrison, S. J., White, P. M., Zock, C., Anderson, 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=Prospective+identification,+isolation+by+flow+cytometry,+and+in+vivo+self-renewal+of+multipotent+mammalian+neural+crest+stem+cells.">Prospective identification, isolation by flow cytometry, and in vivo self-renewal of multipotent mammalian neural crest stem cells.</a> Cell. 96, 737-749 (1999).</li><li>Kruger, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+crest+stem+cells+persist+in+the+adult+gut+but+undergo+changes+in+self-renewal,+neuronal+subtype+potential,+and+factor+responsiveness.">Neural crest stem cells persist in the adult gut but undergo changes in self-renewal, neuronal subtype potential, and factor responsiveness.</a> Neuron. 35, 657-669 (2002).</li><li>Bixby, S., Kruger, G. M., Mosher, J. T., Joseph, N. M., Morrison, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-intrinsic+differences+between+stem+cells+from+different+regions+of+the+peripheral+nervous+system+regulate+the+generation+of+neural+diversity.">Cell-intrinsic differences between stem cells from different regions of the peripheral nervous system regulate the generation of neural diversity.</a> Neuron. 35, 643-656 (2002).</li><li>Walters, L. C., Cantrell, V. A., Weller, K. P., Mosher, J. T., Southard-Smith, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+background+impacts+developmental+potential+of+enteric+neural+crest-derived+progenitors+in+the+Sox10Dom+model+of+Hirschsprung+disease.">Genetic background impacts developmental potential of enteric neural crest-derived progenitors in the Sox10Dom model of Hirschsprung disease.</a> Human Molecular Genetics. , (2010).</li><li>Corpening, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+live+imaging+of+enteric+progenitors+based+on+Sox10-Histone2BVenus+transgene+expression.">Isolation and live imaging of enteric progenitors based on Sox10-Histone2BVenus transgene expression.</a> Genesis. 49, 599-618 (2011).</li><li>Morrison, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Isolation of fetal rat neural crest stem cells (NCSC) from gut and sciatic nerve. , (2012).</li><li>Skarnes, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conditional+knockout+resource+for+the+genome-wide+study+of+mouse+gene+function.">A conditional knockout resource for the genome-wide study of mouse gene function.</a> Nature. 474, 337-342 (2011).</li><li>Harding, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+GUDMAP+database--an+online+resource+for+genitourinary+research.">The GUDMAP database--an online resource for genitourinary research.</a> Development. 138, 2845-2853 (2011).</li><li>Joseph, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enteric+glia+are+multipotent+in+culture+but+primarily+form+glia+in+the+adult+rodent+gut.">Enteric glia are multipotent in culture but primarily form glia in the adult rodent gut.</a> J. Clin. Invest. 121, 3398-3411 (2011).</li><li>Newgreen, D. F., Murphy, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+crest+cell+outgrowth+cultures+and+the+analysis+of+cell+migration.">Neural crest cell outgrowth cultures and the analysis of cell migration.</a> Methods Mol. Biol. 137, 201-211 (2000).</li></ol>

No conflicts of interest declared.

Mouse reporter lines expressing fluorescent reporters are becoming widely available through multiple efforts in the murine genetics community1,8,9. As a result the dissociation method illustrated here can be widely applied for isolation of discrete neuronal subtypes based on neurotransmitter or receptor expression patterns from either fetal or adult tissues. While we have optimized this method based on expression of a fluorescent transgene reporter, it can also be applied to samples expressing cell surface rec...

The authors middle initials were omitted from the publication of <a href="http://www.jove.com/video/4188">An Optimized Procedure for Fluorescence-activated Cell Sorting (FACS) Isolation of Autonomic Neural Progenitors from Visceral Organs of Fetal Mice</a>.

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

<table border="1">
 <tbody>
 <tr>
 <td>Reagent Name</td>
 <td>Vendor</td>
 <td>Catalog number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Accumax</td>
 <td>Sigma(mfr: Innovative Cell Technologies)</td>
 <td>A7089-100ML</td>
 <td>Store frozen in 1 ml aliquots </td>
 </tr>
 <tr>
 <td>DNase I</td>
 <td>Sigma</td>
 <td>D-4527</td>
 <td>Stored frozen at -20 &deg;C 5 mg/ml in 1xHBSS, 
 (Used in Quench, Quench 1:5)</td>
 </tr>
 <tr>
 <td>10X PBS pH 7.4</td>
 <td>Gibco</td>
 <td>70011-044</td>
 <td>Make up to 1x with tissue culture grade water then sterile filter</td>
 </tr>
 <tr>
 <td>10X HBSS w/o Ca or Mg</td>
 <td>Gibco</td>
 <td>14185-052</td>
 <td>Make up to 1x with tissue culture grade water then sterile filter</td>
 </tr>
 <tr>
 <td>Leibovitz's L-15 medium</td>
 <td>Gibco</td>
 <td>21083027</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Penicillin /Streptomycin 100X</td>
 <td>Gibco</td>
 <td>15140-133</td>
 <td>Store aliquoted at -20 &deg;C</td>
 </tr>
 <tr>
 <td>BSA</td>
 <td>Sigma</td>
 <td>A3912-100G</td>
 <td>Store aliquoted at -20 &deg;C, 100 mg/ml in water</td>
 </tr>
 <tr>
 <td>Biowhittaker 1M HEPES in 0.85% NaCl</td>
 <td>Lonza</td>
 <td>17-737E</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>38 &mu;m NITEX Nylon Mesh Membrane</td>
 <td>Sefar America</td>
 <td>3-38/22</td>
 <td>Cut into ~3 cm squares. UV treat overnight to sterilize in the tissue culture hood.</td>
 </tr>
 <tr>
 <td>7-AAD</td>
 <td>Invitrogen</td>
 <td>A1310</td>
 <td>1 mg/ml</td>
 </tr>
 <tr>
 <td>TRIzol LS</td>
 <td>Invitrogen</td>
 <td>10296-028</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>5 ml polystyrene tubes</td>
 <td>Falcon</td>
 <td>352058</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>15 ml conical tubes</td>
 <td>Corning</td>
 <td>430790</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fine Dissecting Forceps</td>
 <td>Fine Science Instruments</td>
 <td>11251-30 </td>
 <td>Dumont#5 forcep, Dumoxel, standard tip 0.1x0.06mm </td>
 </tr>
 <tr>
 <td>Dissecting Spoon</td>
 <td>Fine Science Instruments</td>
 <td>10370-18 </td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about An Optimized Procedure for Fluorescence-activated Cell Sorting FACS Isolation of Autonomic Neural Progenitors from Visceral Organs of Fetal Mice at JoVE.com

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.

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 ...

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Neuroscience

13.7K ビュー.  Vanderbilt University School of Medicine. 胎児のマウス組織からの神経堤由来の神経前駆細胞を精製する最適化された手順が記載されています。このメソッドは、蛍光活性化セルソーティング（FACS）によって離散的集団を分離するために蛍光レポーター遺伝子からの発現を利用しています。技術が開発を通して、大人の組織から神経細胞集団を単離するために適用することができます。

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

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout the developing vertebrate embryo. In addition to providing peripheral glia and neurons, NCC generate melanocytes as well as most of the cranio-facial skeleton. NCC migration and differentiation is controlled by a combination of their axial origin along the neural tube and their exposure to regionally distinct extracellular cues. Such contribution of extracellular ligands is especially evident during the formation of the enteric nervous system (ENS), a complex interconnected network of neural ganglia that locally controls (among other things) gut muscle movement and intestinal motility. Most of the ENS is derived from a small initial pool of NCC that undertake a long journey in order to colonize - in a rostral to caudal fashion - the entire length of the prospective gut. Among several signaling pathways known to influence enteric NCC colonization, GDNF/RET signaling is recognized as the most important. Indeed, spatiotemporally controlled secretion of the RET ligand GDNF by the gut mesenchyme is chiefly responsible for the attraction and guidance of RET-expressing enteric NCC to and within the embryonic gut. Here, we describe an ex vivo cell migration assay, making use of a transgenic mouse line possessing fluorescently labeled NCC, which allows precise quantification of enteric NCC migration potential in the presence of various growth factors, including GDNF.

We present an ex vivo cell migration assay that allows precise quantification of enteric neural crest cell migration potential in the presence of various growth factors.

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

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 ...

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.

We report a Fluorescent Activated Cell Sorting (FACS)-based method to isolate neural stem cells (NSCs) and their progeny from the subventricular zone (SVZ) of the adult mouse brain. Applied to Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) transgenic mice, it allows the study of cell cycle progression by live imaging.

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

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 ...

Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. Additionally, epigenetic chromatin modifications, like histone methylation, have an important role for establishing and maintaining specific cell fates within this process. The vast majority of histone methylation occurs on the flexible histone tail, which is accessible to histone modifiers, erasers, and histone reader proteins. In contrast, H3K79 methylation is located in the globular domain of histone 3 and is implicated in different developmental functions. H3K79 methylation is evolutionarily conserved and can be found in a wide range of species from Homo sapiens to Saccharomyces cerevisiae. The modification occurs in different cell populations within organisms, including neural progenitors. The location of H3K79 methylation in the globular domain of histone 3 makes it difficult to assess. Here, we present methods to isolate and culture cortical progenitor cells (CPCs) from embryonic cortical brain tissue (E11.5-E14.5) or cerebellar granular neuron progenitors (CGNPs) from postnatal tissue (P5-P7), and to efficiently immunoprecipitate H3K79me2 for quantitative PCR (qPCR) and genome-wide sequencing.

We present an effective and reproducible method to isolate and culture neural progenitor cells from embryonic and postnatal brain tissue for chromatin immunoprecipitation (ChIP) of histone 3 lysine 79 dimethylation (H3K79me2) - a histone mark located within the globular domain of histone 3.

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. ...

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 ...

Dissection of Pelvic Autonomic Ganglia and Associated Nerves in Male and Female Rats

The bilateral major pelvic ganglia (MPG; synonym, pelvic ganglia) are the primary source of postganglionic sympathetic and parasympathetic neurons innervating pelvic organs of rodents; the functionally equivalent structure in humans is the inferior hypogastric plexus. The major pelvic ganglia also provide the route by which lumbar and sacral sensory axons reach the pelvic organs. These complex, mixed ganglia can prove challenging to identify and dissect for further experimental study of normal autonomic mechanisms or to establish preclinical models of disease, injury or visceral pain. Here we describe a protocol to access and visualize these ganglia and their associated nerve tracts. We provide this protocol with schematics for both male and female rats, as the ganglion size and landmarks for identification differ between sexes. The protocol describes removal of the ganglion for in vitro studies, but this method can be integrated into a surgical recovery protocol for experimental interventions (e.g., nerve crush, nerve resection) or for mapping neuronal circuits (e.g., by microinjection of neural tracers). We also demonstrate the primary structures of the ganglion and its associated nerves immediately following dissection and following immunohistochemical staining.

The major pelvic ganglia contain parasympathetic and sympathetic neurons that innervate pelvic organs. Here we describe a dissection method and provide schematics for identification of these ganglia and their associated nerves. These methods can be applied to experimental manipulation of these ganglia in vivo or removal post-mortem for further study.