Wir bedanken uns bei Jonna Ellis für die redaktionelle Unterstützung. Vom National Institute of Mental Health: Das Projekt beschrieben wurde von Verleihungsnummer R01MH079751 (F. Peruzzi PI) unterstützt. Der Inhalt ist ausschließlich der Verantwortung der Autoren und nicht unbedingt die offizielle Meinung des National Institute of Mental Health oder des National Institutes of Health.

1. Poly-D-Lysin (PDL): Herstellung <ol><li> 5 ml sterilem ddH 2 O bis 5 mg PDL um eine Stammlösung von 1 mg / ml zu erhalten. </li><li> Mischen Sie Stammlösung durch mehrmaliges Pipettieren. </li><li> Sofort verwenden oder speichern Poly-D-Lysin-Lösung bei 2-8 ° C. </li></ol> 2. Poly-D-Lysin (PDL): Beschichtung Zellkulturschalen <ol><li> Verdünnen Sie die PDL-Stammlösung mit sterilem ddH 2 O auf die Endkonzentration von 10 ug / ml. </li><li> Pipette genug Lösung in eine 60 mm-Schale zur Kultur-Oberfläche (3 ml für eine 60 mm-Schale) zu decken. </li><li> Schaukeln sanft auf gleichmäßige Beschichtung der Oberfläche Kultur zu gewährleisten. </li><li> Inkubieren beschichtete Platten bei Raumtemperatur (RT) über Nacht. </li><li> Am nächsten Tag, in der Regel der Tag der Dissektion, entfernen Sie das Poly-D-Lysin-Lösung durch Absaugen und waschen kurz mit 3 ml sterilem ddH 2 O. Wiederholen Sie diesen Schritt. Nach dem zweiten Waschen, Wasser vollständig zu entfernen durch Absaugen. </li><li>Die Platten können bei 4 ° C für bis zu drei Wochen gelagert werden. </li></ol> 3. Poly-D-Lysin (PDL) und Laminin: Vorbereitung und Beschichtung von Glas Zwei-Kammer-Objektträgern <ol><li> Mix PDL (1 mg / ml) und Laminin (1 mg / ml) Stammlösungen in sterilem ddH 2 O bis zu einer Endkonzentration von 10 und 5 g / ml. </li><li> Pipette genug Lösung in die Vertiefungen einer Glas Zwei-Kammer-Objektträger, die Kultur Oberfläche (1 ml pro Vertiefung einer 2 gut Glas Zwei-Kammer-Objektträger) bestimmt. </li><li> Schaukeln sanft auf gleichmäßige Beschichtung der Oberfläche Kultur zu gewährleisten. </li><li> Inkubieren beschichteten Platten über Nacht bei RT. </li><li> Am nächsten Tag, entfernen Sie das Poly-D-Lysin-Beschichtung Laminin-Lösung durch Absaugen und waschen Sie kurz zweimal mit 1 ml sterilem ddH 2 O. Nach dem zweiten Waschen, Wasser vollständig zu entfernen durch Absaugen. </li><li> Chamber Slides können bei 4 ° C für bis zu drei Wochen gelagert werden. </li></ol> Hinweis: Jeder Glas Chamber Slide können beschichtet werden following dieses Protokoll. Wir verwenden oft die Zwei-Kammer-Objektträgern, weil jede Folie ermöglicht die Steuerung-Test experimentellen Setting (z. B. im Vergleich zu unbehandelten behandelt, im Vergleich zu untransfizierte transfiziert). 4. Neuronale Dissection und Kultur <ol><li> Wärmen Sie die folgenden Reagenzien in einem 37 ° C warmes Wasserbad: <ul type="disc"><li> TrypLE Express auf seiner ursprünglichen 100 ml Flasche. </li><li> Neurobasal/B27 Vollmedium (siehe Tabelle I). Das Volumen erwärmt hängt von der Anzahl der Gänge zu plattierenden (zB 30 ml für zehn 60 mm beschichtet Geschirr). </li></ul></li><li> 3 ml kaltem Ruhezustand E-Lösung, um vier 60 mm-Kulturschalen und 13 ml einer 15 ml Falcon BD hohe Klarheit Polypropylen konische Röhre. </li><li> In 25-30 ml kaltem Dissektion Medium (siehe Tabelle II, Dr. Olimpia Meucci, persönliche Mitteilung) zu jeder der drei 100 mm-Kulturschalen. Diese Platten, die eine große Volumen des Mediums, wird verwendet, um die Embryonen unmittelbar nach ihrer waschenEntnahme aus den Fruchtblasen (Schritte 4.7 und 4.8). </li><li> Euthanize einen E17 timed trächtigen Ratten durch CO 2 in Übereinstimmung mit Public Health Services Policy über humane Pflege und Verwendung von Labortieren und unter einer institutionell genehmigt Tierpflege und nutzen Protokoll. </li><li> Sprühen Sie Unterbauch mit 70% EtOH und schneiden medial durch die Haut und Muskeln mit einer Schere Aussetzen der Gebärmutter und Embryonen. </li><li> Entfernen Sie alle Föten und legen Sie sie in einem sterilen 100-mm-Schale mit einem Überschuß von kaltem Medium Dissektion (25-30 ml, siehe Schritt 4.3). </li><li> Schneiden Embryonen mit Hilfe eines kleinen Schere aus Fruchtblase und legen Sie sie in der zweiten 100-mm-Schale mit kaltem Medium Dissektion. </li><li> Waschen der Embryonen bei Raumtemperatur durch leichtes Kippen des 100-mm-Schale für 5-10 Sekunden. Dann übertragen Sie die gespülten Embryonen in die dritte Schale mit 100 mm Sezieren Medium. Zwei Waschungen in überschüssige Medium sind in der Regel aus, um alle Spuren von Blut zu entfernen. Jedoch, wenn notwendig,Essary, waschen Sie ein weiteres Mal mit einer frischen 100-mm-Schale mit 25-30 ml kaltem Medium Dissektion. </li><li> Mit einem Stereomikroskop und einer gebogenen Pinzette, extrahieren jeder Ratte Embryo das Gehirn durch Herausziehen der Haut und Schädel. Zeigen gesamte Gehirn in einem der 60-mm-Schalen (in der Regel mit nicht mehr als 5 Gehirne pro Schale) mit kaltem Hibernate E. Bewahren Sie diese Platten auf Eis. </li><li> Nehmen Sie eine Schüssel zu einer Zeit und unter einem Binokular, trennen Sie die Hemisphären und isolieren die zerebralen Kortex Entfernen des Mittelhirns und der Hirnhäute. </li><li> Optional: Schnitt Gehirne auf die Mittellinie, extrahieren hippocampi, und gehen Sie folgendermaßen vor, um Hippocampus-Neuronen zu isolieren. </li><li> Sammeln Sie alle seziert Kortex in einem 15-ml klare konischen Rohr mit 13 ml kaltem Hibernate E. Lassen Sie die zerebralen Kortex auf Eis, bis alle Sektionen abgeschlossen sind. Aufgrund ihrer geringen Größe kann präpariert hippocampi in einem 1,5-ml-Eppendorf-Röhrchen anstelle einer 15-ml-Röhrchen gesammelt werden. Falls gewünscht, in diesem Schritt Kortex oderhippocampi in einem Kryoröhrchen Fläschchen mit 1 ml gebracht werden Ruhezustand E + B27 + 2% Gentamicin (50 ug / ml) + Fungizone (250 ng / ml) im Verhältnis von 2-4 oder 2-4 Kortex hippocampi pro Röhrchen. Hirngewebe bei 4 ° C im Dunkeln bis zu einer Woche (späteren Zeiten nicht noch nicht getestet) gespeichert werden. Bei Bedarf nutzen, um die feinen Pinzette Hirngewebe in ein 15 ml Röhrchen mit Hibernate E übertragen und folgen Sie dann das Protokoll unten, um Neuronen zu isolieren. </li><li> Übertragen der Röhre eine Gewebekulturabzug. Lassen Sie die Rinden auf dem Boden des Röhrchens absetzen und dann vorsichtig den Überstand. </li><li> In 13 ml frisch Hibernate E an den 15-ml konischen Röhrchen, erlauben Kortex am Boden des Röhrchens absetzen und entfernen Sie vorsichtig den Überstand. Wiederholen Sie diesen Schritt 2 weitere Male und nach dem letzten Waschen, sorgfältig entfernen Sie alle Medien. </li><li> Enzymatisch verdauen zerebralen Kortex mit indem 1-2 ml (abhängig von der Anzahl Rindenanzahl; verbrauchen weniger für hippocampi Trennung) von warmem TrypLE Express. Verschließen Sie den Deckel des Röhrchens mit Parafilm und schweben Sie den Schlauch in einem 37 ° C warmen Wasserbad für 10 Minuten. </li><li> Sprühen Sie das Rohr mit 70% Ethanol vor dem Öffnen der Kappe und 10 ml Hibernate E. Lassen Sie die Rinden am Boden des Röhrchens absetzen und den Überstand entfernen. Wiederholen Sie diesen Schritt dreimal zu waschen TrypLE Express. Im letzten Schritt entfernen Sie vorsichtig alle Medien. </li><li> Sanft verreiben (4-5 mal) die Rinden in 2 ml Neurobasal/B27 Vollmedium mit einem Feuer-poliertem Glas Pasteur (ca. 1 mm im Durchmesser). Seien Sie vorsichtig, um Blasen zu vermeiden. </li><li> Wiederholen weitere 4-5 Mal mit einer sterilen Pasteurpipette aus Glas kleineren Durchmesser (dh eine Pipette zu 1/2-3/4 mm im Durchmesser). Verwenden Sie nicht einer Pasteurpipette kleiner als diese, oder es wird die Zellen zu zerstören. </li><li> Erlauben restlichen Stücke von Gewebe (in der Regel nur sehr wenige, wenn überhaupt) zu begleichen. </li><li> Übertragen der oberen Einzelzell-Suspension auf ein neues 15-ml-Röhrchen, hinterlässt ständiger Gewebestücke. Pelzther verdünnen die Zellsuspension bis zu 10-12 ml mit Neurobasal/B27 Vollmedium. </li><li> Gut mischen und verdünnen Zellen zum Zählen durch Zugabe von 10 ul Zellsuspension zu 490 ul 50x Counting Lösung (siehe Tabelle III) in einem 1,5 ml Eppendorf-Röhrchen. </li><li> Tafel Zellen auf PDL-beschichteten Platten mit einer Dichte von 5,0 x 10 4 / cm 2. Wenn Nukleofektion durchgeführt werden soll, empfehlen wir Ausplattieren der Zellen in einer höheren Konzentration (8-10 x 10 4 / cm 2). </li><li> Normalerweise sezieren wir 9-10 Feten pro Experiment, wie etwa 13 x 10 6 Neuronen aus jedem E17 Fötus stammen. Wenn mehr Embryonen benötigt werden, um sicherzustellen, dass die ganze Prozedur dauert nicht länger als zwei Stunden. </li><li> Falls gewünscht, 24 Stunden nach der Isolierung können 10 uM Cytosin-β-D-arabinofuranosid (AraC) jeder Schale zugegeben werden, um Gliawucherung verhindern. Jedoch ist dieser Schritt nicht erforderlich, da Neurobasal/B27 Medium inhibiert Gliawucherung, Gemäß den Empfehlungen des Herstellers (Invitrogen / Gibco). </li><li> Neuronen können für Experimente nach 4-5 Tagen in vitro verwendet werden, obwohl genaue Zeit hängt von der gewünschten Differenzierung der Bühne. Wir haben kultivierten Neuronen für bis zu 4 Wochen ohne eine signifikante Abnahme der Überlebensrate (Abbildung 1). </li><li> Für längere Kultivierung, ersetzen Kulturmedium jede Woche mit frisch zubereiteten Neurobasal/B27 Vollmedium. </li></ol> 5. Repräsentative Ergebnisse Neuronen auf Glas Chamber Slides kultiviert werden können, um Immunzytochemie unterzogen werden. 1 zeigt ein typisches Bild einer kortikalen Neuronen nach fünf Tagen in Kultur fixiert und immunmarkiert mit anti-MAP-2-Antikörper auf neuronale Prozesse zeigen. 2 zeigt ein repräsentatives Bild einer Ratte Hippocampus nach 3 Wochen in Kultur. Die neuronale Morphologie einer ausdifferenzierten Zelle wird durch MAP-2 hervorgehoben immunolabeling (MAP-2 neuronalen Marker, monoklonalen Maus-Antikörper Klon AP-20, Gene Tex, Irvine, CA) nach einem Standard-Verfahren wie zuvor 1 beschrieben. Die Bilder wurden sichtbar gemacht mit der Nikon Eclipse E400 aufrecht Fluoreszenz-Mikroskop mit Kamera EXI aqua (Qimaging), motorisierte Z-Achse, und SlideBook5 Akquisition / Dekonvolution Software (Intelligent Imaging Innovations, Inc., Denver, CO) ausgestattet. Eine Reihe von dreidimensionalen Bildern der einzelnen Bild wurden zu einem zweidimensionalen Bild zerlegt und aufgelöst durch Anpassung der Signal Cut-off in der Nähe von maximaler Intensität, um die Auflösung zu erhöhen. Abbildung 3 zeigt Reinheit der neuronalen Kulturen. Protein wurden Lysate DIV7 Ratte neuronalen Kulturen (CTX) und von einem Fall von humanen Glioblastomen (GBM) erhalten. Wie erwartet, ist die neuronale Lysat stark positiv für die neuronale Protein MAP-2 und negativ für den Astrozyten-Marker GFAP, während der GBM Proteinlysatpräparation negativ ist foder MAP-2 und positiv für GFAP. Obwohl in unserem Protokoll wir verwendet haben Hibernate E für mehrere Jahre als sezieren und Spülmedium, vor kurzem haben wir ein zusätzliches und sehr praktischen Gebrauch davon, um Hirngewebe zur weiteren Verwendung zu erhalten erforscht. Abbildung 4 zeigt einen Tag in-vitro-5 (DIV5) Kultur von Ratten-kortikalen Neuronen aus Kortex bei 4 ° C für eine Woche lang in den Ruhezustand E + B27 ihres ursprünglichen Präparation von den Embryonen isoliert. Neuronen wurden auf einer Glasbasis Zwei-Kammer-Objektträger mit PDL und Laminin beschichtet, wie zuvor beschrieben ausplattiert. Das aufgenommene Bild wurde mit Hilfe entfaltet SlideBook5 Akquisition / Dekonvolution Software wie oben beschrieben (Abb. 2). <img alt="1" src="/files/ftp_upload/3965/3965fig1.jpg" /> Abbildung 1. Repräsentatives Bild einer kortikalen Neuronen mit pmaxGFP (Amaxa, Lonza, Walkersville, MD) und nucleofected immunmarkiert mit MAP-2 Antikörper, in rot. Original-Vergrößerung 100x. <img alt="2" src="/files/ftp_upload/3965/3965fig2.jpg" /> Abbildung 2. Vertreter Bild zeigt MAP-2 Immunomarkierung, in rot, von Neuronen im Hippocampus nach 3 Wochen in der Kultur. DAPI-Färbung, in blau, zeigt Zellkernen. Original-Vergrößerung 40x. <img alt="Abbildung 3" src="/files/ftp_upload/3965/3965fig3.jpg" /> 3. Western-Blot, Reinheit von neuronalen Zellkulturen. 30 ug neuronalen Ratten und menschlichen GBM Protein-Lysate wurden durch Elektrophorese aufgetrennt und einer Western-Blot-Analyse nach Standardverfahren ein. Anti-MAP-2 war ein polyklonaler Hasen von Cell Signaling (Danvers, MA), wurde Anti-GFAP-Antikörper ein monoklonaler Maus von Chemicon (Millipore, Billerica, MA), und die monoklonalen anti-GRB2 Antikörper wurde von Transduction Laboratories BD ( Sparks, MD). GRB2 wurde als Kontrolle verwendet Belastung. </p&gt; <img alt="Abbildung 4" src="/files/ftp_upload/3965/3965fig4.jpg" /> Abbildung 4. Vertreter Bilder der Tage in vitro 5 (DIV5) kortikale Neuronen der Ratte aus Kortex links in Hibernate erhaltenen E + B27 bei 4 ° C für eine Woche nach ihrer Präparation. A) Phasenkontrast von Neuronen auf einem Glas-Zwei-Kammer-Objektträger kultiviert. Original-Vergrößerung 20x. B) Immunfluoreszenz, der die Expression von MAP-2 bei der neuronalen Prozessen, grünen, die Kultur wurde für die Astrozyten-Marker GFAP negativ. DAPI-Färbung, in blau, zeigt Zellkernen. Original-Vergrößerung 40x.

<ol>
	<li>Aprea, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tubulin-mediated+binding+of+human+immunodeficiency+virus-1+Tat+to+the+cytoskeleton+causes+proteasomal-dependent+degradation+of+microtubule-associated+protein+2+and+neuronal+damage.">Tubulin-mediated binding of human immunodeficiency virus-1 Tat to the cytoskeleton causes proteasomal-dependent degradation of microtubule-associated protein 2 and neuronal damage.</a> J. Neurosci. 26, 4054-4062 (2006).</li><li>Kivell, B. M., McDonald, F. J., Miller, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum-free+culture+of+rat+post-natal+and+fetal+brainstem+neurons.">Serum-free culture of rat post-natal and fetal brainstem neurons.</a> Brain Res. Dev. Brain Res. 120, 199-210 (2000).</li><li>Kivell, B. M., McDonald, F. J., Miller, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+serum-free+culture+of+late+fetal+and+early+postnatal+rat+brainstem+neurons.">Method for serum-free culture of late fetal and early postnatal rat brainstem neurons.</a> Brain Res. Brain Res. Protoc. 6, 91-99 (2001).</li><li>Brewer, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum-free+B27/neurobasal+medium+supports+differentiated+growth+of+neurons+from+the+striatum,+substantia+nigra,+septum,+cerebral+cortex,+cerebellum,+and+dentate+gyrus.">Serum-free B27/neurobasal medium supports differentiated growth of neurons from the striatum, substantia nigra, septum, cerebral cortex, cerebellum, and dentate gyrus.</a> J. Neurosci. Res. 42, 674-683 (1995).</li><li>Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culturing nerve cells. , (1998).</li><li>Eletto, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+SNAP25+expression+by+HIV-1+Tat+involves+the+activity+of+mir-128a.">Inhibition of SNAP25 expression by HIV-1 Tat involves the activity of mir-128a.</a> J. Cell Physiol. 216, 764-770 (2008).</li><li>Gualco, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IGF-IR-dependent+expression+of+Survivin+is+required+for+T-antigen-mediated+protection+from+apoptosis+and+proliferation+of+neural+progenitors.">IGF-IR-dependent expression of Survivin is required for T-antigen-mediated protection from apoptosis and proliferation of neural progenitors.</a> Cell Death Differ. 17, 439-451 (2010).</li><li>Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+and+differentiation+of+adult+neuronal+progenitor+cells+transplanted+to+the+adult+brain.">Survival and differentiation of adult neuronal progenitor cells transplanted to the adult brain.</a> Proc. Natl. Acad. Sci. U.S.A. 92, 11879-11883 (1995).</li><li>Keyser, D. O., Pellmar, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+transmission+in+the+hippocampus:+critical+role+for+glial+cells.">Synaptic transmission in the hippocampus: critical role for glial cells.</a> Glia. 10, 237-243 (1994).</li><li>Pfrieger, F. W., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+efficacy+enhanced+by+glial+cells+in+vitro.">Synaptic efficacy enhanced by glial cells in vitro.</a> Science. 277, 1684-1687 (1997).</li></ol>

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Die Methode der Präparation und Kultur von Ratten-Hippocampus und kortikalen Neuronen hier beschriebenen ermöglicht die Durchführung von Experimenten unter Verwendung nahezu reinen neuronalen Kulturen in einem chemisch definierten Medium (Abbildung 3) gewachsen. Obwohl Protokolle für die Kultivierung nahezu reinem Neuronen in serumfreiem Medium wurden zuvor 2,3,4 beschrieben, gibt es wesentliche Änderungen in unserer Methode. Anders als bei traditionellen Protokollen (zB Banker et al.) <...

<table border="1"><tbody><tr><td> Reagens </td><td> Konzentration </td></tr><tr><td> Neurobasal </td><td> 98% </td></tr><tr><td> B27 </td><td> 2% </td></tr><tr><td> Glutamax </td><td> 0,5 mM </td></tr></tbody></table> Tabelle I Neurobasal/B27 Vollmedium. <table border="1"><tbody><tr><td> Reagens </td><td> Konzentration </td></tr><tr><td> Glucose </td><td> 16 mM </td></tr><tr><td> Saccharose </td><td> 22 mM </td></tr><tr><td> HEPES </td><td> 10 mM </td></tr><tr><td> NaCl </td><td> 160 mM </td></tr><tr><td> KCl </td><td> 5 mM </td></tr><tr><td> Na 2 HPO 4 </td><td> 1 mM </td></tr><tr><td> KH 2 PO 2 </td><td> 0,22 mM </td></tr><tr><td> Gentamicin </td><td> 50 ug / ml </td></tr&gt; <tr><td> Fungizone </td><td> 250 ng / ml </td></tr><tr><td> pH-Wert </td><td> 7,4 </td></tr><tr><td> Osmolarität </td><td> 320-330 mOsm </td></tr></tbody></table> Tabelle II. Dissection Medium. <table border="1"><tbody><tr><td> Reagens </td><td> Volume (ul) </td></tr><tr><td> Neurobasal/B27 Vollmedium </td><td> 240 </td></tr><tr><td> Trypanblau Stain 0,4% </td><td> 250 </td></tr><tr><td> Gesamt </td><td> 490 </td></tr></tbody></table> Tabelle III. 50x Counting Lösung. <table border="1"><tbody><tr><td> Reagens </td><td> Firma </td><td> Cat. Anzahl </td></tr><tr><td> Hibernate E </td><td> Brainbits </td><td> 767171 </td></tr><tr><td> Neurobasal </td><td> Gibco, Invitrogen </td><td> 21103-049 </td></tr><tr><td> B27 </td><td> Gibco, Invitrogen </td><td> 17504-044 </td></tr><tr><td> Fungizone </td><td> Gibco, Invitrogen </td><td> 15290-018 </td></tr><tr><td> Gentamicinsulfat </td><td> Sigma Aldrich </td><td> G1264 </td></tr><tr><td> 200 mM Glutamax </td><td> Gibco, Invitrogen </td><td> 35050 </td></tr><tr><td> TrypLE Express w / o Phenolrot </td><td> Gibco, Invitrogen </td><td> 12604 </td></tr><tr><td> Cytosin-β-D-Hydrochlorid arabinofuranosid </td><td> Sigma Aldrich </td><td> C6645 </td></tr><tr><td> Poly-D-Lysin </td><td> Sigma Aldrich </td><td> P6407 </td></tr><tr><td> Laminin 1 mg / ml </td><td> Millipore </td><td> CC095 </td></tr><tr><td> HEPES </td><td> Sigma Aldrich </td><td> H3375 </td></tr><tr><td> Trypanblau Stain 0,4% </td><td> Gibco, Invitrogen </td><td> 15250 </td></tr></tbody></table&gt; Tabelle IV. Spezifische Reagenzien. <table border="1"><tbody><tr><td> Ausrüstung </td><td> Firma </td><td> Cat. Anzahl </td></tr><tr><td> Stereo-Mikroskop </td><td> Olymp </td><td> SZ61 </td></tr><tr><td> Große Zange </td><td> FST </td><td> 11022-14 </td></tr><tr><td> Fine-Pinzetten </td><td> Moria </td><td> MC40B </td></tr><tr><td> Micro Fine-Pinzetten </td><td> Moria </td><td> MC31 </td></tr><tr><td> Gestochen scharfe Schere </td><td> Roboz </td><td> RS-6820 </td></tr><tr><td> Micro Präparierschere </td><td> FST </td><td> 91460-11 </td></tr><tr><td> Micro Dissecting gebogenen Schere </td><td> FST </td><td> 14067-11 </td></tr><tr><td> Glas 2-Kammer-Objektträgern </td><td> Lab-Tek </td><td> 154461 </td></tr><tr><td> 60 mm Platten </td&gt; <td> BD Falcon </td><td> 353002 </td></tr><tr><td> 100 mm-Schalen </td><td> Corning </td><td> 430167 </td></tr><tr><td> 15 ml Röhrchen </td><td> BD Falcon </td><td> 352099 </td></tr><tr><td> 1,5 ml Kryo-Röhrchen Fläschchen </td><td> Nunc </td><td> 375353 </td></tr></tbody></table> Tabelle V. Ausstattung.

isolation and culture of rat embryonic neural cells: a quick protocol

Wir beschreiben eine schnelle Methode zur Dissoziation und Kultur oder kortikalen Neuronen des Hippocampus von E15-17 Rattenembryonen. Das Verfahren kann erfolgreich zur Isolierung von Maus und Mensch primären Neuronen und neuralen Vorläuferzellen angewendet werden. Dissoziierte Neuronen in serumfreiem Medium bis zu mehreren Wochen aufrechterhalten. Diese Kulturen können Nukleofektion, Immunzytochemie, Nukleinsäuren Herstellung sowie Elektrophysiologie verwendet werden. Ältere neuronalen Kulturen können auch mit einem guten Wirkungsgrad von lentiviralen Transduktion und, weniger effizient, mit Calciumphosphat oder Lipid-basierte Methoden wie Lipofectamin transfiziert werden.

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Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

Wir beschreiben eine schnelle Methode zur Isolierung und Kultur hippokampalen und kortikalen Neuronen aus Nagetier Embryonen. Dieses Protokoll erlaubt es uns, Experimente, in denen nahezu reinen neuronalen Kulturen sind erforderlich, durchzuführen.

Isolierung und Kultivierung von embryonalen Ratten-Nervenzellen: ein Quick-Protokoll

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

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38.0K Aufrufe.  LSU Health Sciences Center - New Orleans. Wir beschreiben eine schnelle Methode zur Isolierung und Kultur hippokampalen und kortikalen Neuronen aus Nagetier Embryonen. Dieses Protokoll erlaubt es uns, Experimente, in denen nahezu reinen neuronalen Kulturen sind erforderlich, durchzuführen.

Serie: Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Dissection und Kultur kommissuralen Nervenzellen aus embryonalen Rückenmark

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

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Neurowissenschaften

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.
This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

Isolation und Expansion der erwachsenen Maus Neural Stem Cells mit dem Neurosphäre Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

Gründung Embryonale Maus Neural Stem Cell Culture Mit dem Neurosphäre Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells1. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells2, melanocyte stem cells3 and neural crest like stem cells4-7. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles4,5. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury8. Furthermore, peripheral nerves have been repaired with stem cell grafts9, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination10. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models.
Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.

Isolation und Kultur von Neural Crest Stem Cells aus menschlichen Haarfollikeln

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. Neuroepithelial cells of the neural tube that express Nkx6.1 are the unique precursor cells for spinal motoneurons1. Though postmitotic motoneurons move towards their final position and organize themselves into columns along the spinal tract2,3. More than 90% of all these differentiated and positioned motoneurons express the transcription factors Islet 1/2. They innervate the muscles of the limbs as well as those of the body and the inner organs. Among others, motoneurons typically express the high affinity receptors for brain derived neurotrophic factor (BDNF) and Neurotrophin-3 (NT-3), the tropomyosin-related kinase B and C (TrkB, TrkC). They do not express the tropomyosin-related kinase A (TrkA)4. Beside the two high affinity receptors, motoneurons do express the low affinity neurotrophin receptor p75NTR. The p75NTR can bind all neurotrophins with similar but lower affinity to all neurotrophins than the high affinity receptors would bind the mature neurotrophins. Within the embryonic spinal cord, the p75NTR is exclusively expressed by the spinal motoneurons5. This has been used to develop motoneuron isolation techniques to purify the cells from the vast majority of surrounding cells6. Isolating motoneurons with the help of specific antibodies (panning) against the extracellular domains of p75NTR has turned out to be an expensive method as the amount of antibody used for a single experiment is high due to the size of the plate used for panning. A much more economical alternative is the use of lectin. Lectin has been shown to specifically bind to p75NTR as well7. The following method describes an alternative technique using wheat germ agglutinin for a preplating procedure instead of the p75NTR antibody. The lectin is an extremely inexpensive alternative to the p75NTR antibody and the purification grades using lectin are comparable to that of the p75NTR antibody. Motoneurons from the embryonic spinal cord can be isolated by this method, survive and grow out neurites.

An alternative way of isolating mouse embryonic motoneurons from the spinal cord is described. The method takes into account the fact that lectin can bind to the low affinity nerve growth factor receptor p75NTR. This lectin-based preplating allows a purification similar to that with a specific antibody against the p75NTR.

Lectin-basierte Isolierung und Kultivierung von embryonalen Maus-Motoneuronen

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. ...

Isolation and Culture of Human Fungiform Taste Papillae Cells

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple stimuli and complex chemical molecules contained in foods. In human, individual fungiform papillae contain from zero to as many as 20 taste buds. There is no established protocol for culturing human taste cells, although the ability to maintain taste papillae cells in culture for multiple cell cycles would be of considerable utility for characterizing the molecular, regenerative, and functional properties of these unique sensory cells. Earlier studies of taste cells have been done using freshly isolated cells in primary culture, explant cultures from rodents, or semi-intact taste buds in tissue slices1,2,3,4. Although each of these preparations has advantages, the development of long-term cultures would have provided significant benefits, particularly for studies of taste cell proliferation and differentiation. Several groups, including ours, have been interested in the development and establishment of taste cell culture models. Most attempts to culture taste cells have reported limited viability, with cells typically not lasting beyond 3-5 d5,6,7,8. We recently reported on a successful method for the extended culture of rodent taste cells9. We here report for the first time the establishment of an in vitro culture system for isolated human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability. Cells displayed many molecular and physiological features characteristic of mature taste cells. Gustducin and phospholipase C &beta;2, (PLC-&beta;2) mRNA were detected in many cells by reverse transcriptase-polymerase chain reaction and confirmed by sequencing. Immunocytochemistry analysis demonstrated the presence of gustducin and PLC-&beta;2 expression in cultured taste cells. Cultured human fungiform cells also exhibited increases in intracellular calcium in response to appropriate concentrations of several taste stimuli indicating that taste receptors and at least some of the signalling pathways were present. These results sufficient indicate that taste cells from adult humans can be generated and maintained for at least eight passages. Many of the cells retain physiological and biochemical characteristics of acutely isolated cells from the adult taste epithelium to support their use as a model taste system. This system will enable further studies of the processes involved in proliferation, differentiation and function of mammalian taste receptor cells in an in vitro preparation.
Human fungiform taste papillae used for establishing human fungiform cell culture were donated for research following proper informed consent under research protocols that were reviewed and approved by the IRB committee. The protocol (#0934) was approved by Schulman Associates Institutional Review Board Inc., Cincinnati, OH. Written protocol below is based on published parameters reported by Ozdener et al. 201110.

We aimed to develop a reproducible protocol for isolating and maintaining long-term cultures of human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability.

Isolierung und Kultivierung humaner Zellen pilzförmige Papillen Geschmack

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple ...

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.

Isolierung und Kultivierung von Zellen der Neuralleiste aus embryonalen murinen Neuralrohr

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

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and M&uuml;ller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18.
While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45.
Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,44-45.
However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

Dissection, Kultur und Analyse der<em&gt; Xenopus laevis</em&gt; Embryonic Netzhautgewebe

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

Isolierung und Kultivierung von Endothelzellen aus dem embryonalen Vorderhirn

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

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.

Eine Maus, zwei Kulturen: Isolierung und Kultur von adulten neuralen Stammzellen aus dem Zwei Neurogene Zonen der einzelnen Mäuse

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