Diese Studie wurde teilweise durch eine Anschubfinanzierung von der Texas A &amp; M University finanziert

Vorbereitung vor der Dissektion Chirurgische Instrumente: sterilisieren alle Dissektion Schere und Pinzette von Autoklaven Deckglas Reinigung: <ol><li> Weichen Sie alle Deckgläser (15mm im Durchmesser für 24-Well-Platte) in 1L Gras Becherglas in 33% HCl für mindestens 24 h </li><li> Waschen mit fließendem Wasser für 10 min mit gelegentlichen schütteln, um alle verbleibenden HCl zu entfernen </li><li> Spülen mit Millipore-Wasser </li><li> Ablassen von Wasser aus </li><li> Soak Deckgläser in 95-98% Ethanol </li><li> Transfer Deckgläser zu einem sauberen Tuch auf einem flachen Teller und an der Luft trocknen die Deckgläser </li><li> Transfer Deckgläschen in ein Becherglas, mit Alufolie abdecken und Autoklaven Hinweis: Deckgläser kann in diesem Stadium gespeichert werden </li><li> Legen Sie individuelle Deckglas in die Kultur-Platten. Bereit für die Beschichtung </li></ol> Deckglas-Beschichtung: <ol><li> Verdünnen Sie 100 x Lager von Poly-D-Lysin (PDL, 10 mg / ml in 0,5% Rinderserumalbumin in PBS, als Aliquots bei -20 ° C gelagert) mit PBS und Filter-sterilisiert (0,22 um) </li><li> Coat Deckgläser mit 1 x PDL-Lösung (~ 0,15 ml / cm 2) für 2 h bei 37 ° C im Brutschrank </li><li> Entfernen PDL-Lösung und 3 x waschen mit sterilem ddH 2 O und trocken Deckgläser komplett </li></ol> Hinweis: Coat genug Deckgläser / Platten für jede Sektion. PDL-beschichtete Deckgläser kann für ein paar Wochen bei 4 ° C gelagert werden Medien und Lösungen Dissection (DM): Verwenden Sie sterile eiskalt Hanks Balanced Salt Solution (w / o Ca 2 +, Mg 2 +) (HBSS, Invitrogen 14175) ergänzt mit 10 mM HEPES (Invitrogen 15630). Aggregate Kulturmedien: Die DMEM/NBB27 Medium enthält DMEM / Neurobasal (1:1 vol: vol), 2% B27, 1 x Sato, 0,5 mM Natriumpyruvat, 0,75 Glutamax, 60μg/ml N-Acetylcystein, 5 ug / ml Insulin, 10nm d-Biotin und 1% Penicillin / Streptomycin. Um 100 ml des Aggregats Kulturmedium mischen 50 ml DMEM (w / o Pyruvat / Glutamin, Invitrogen 11960), 50 ml Neurobasal Medium (Invitrogen 211034), 375 ul Glutamax (100 x, Invitrogen 35.050), 5,5 mg Natrium-Pyruvat (Sigma p2256), 2 ml B27 (Invitrogen 17504), 6,3 mg N-Acetylcystein (Sigma A8199), 1 ml Sato Lager (100 x Lager, siehe unten), 25 ul d-Biotin Lager (4000 x Lager, 40 uM in PBS als Aliquots bei -20 ° C. Sigma B4639), 100 ul von Insulin (1000 x Lager, 5 mg / ml in 0,01 N HCl als Aliquots bei -20 ° C gelagert, Sigma 16634) und 1 ml Penicillin gespeichert / Streptomycin (100 x Lager, Invitrogen 15140), Filter-sterilisiert und bei 4 ° C. Sato 100 x Stammlösung: Zu 40 ml der zu machen: Mix 40 ml Neurobasal mit 400 mg apo-Transferrin (Sigma T2252), 400 mg BSA (Sigma A9647), 10 ul von Progesteron (25 mg / ml Ethanol, als gespeicherte Aliquots bei -20 ° C. Sigma P8783), 64 mg Putrescin (Sigma P7505) und 40 ul von Natriumselenit (30 nM in PBS, als Aliquots bei -20 ° C gelagert, Sigma S5261). Filter sterilisieren Sato Stammlösung und speichern als Aliquots bei -20 ° C. Neuron plating Medium (PM): Neurobasalmedium, 2% B27, 2 mM Glutamin (100x Lager, Aliquots bei -20 ° C, Invitrogen 25030 gespeichert), 25 um Glutaminsäure (100 Aktien, bei -20 ° C gelagert) und 1% Penicillin / Streptomycin. Neuron Kulturmedium (CM): Neurobasalmedium, 2% B27, 2 mM Glutamin (100x Lager, Aliquots bei -20 ° C gelagert) und 1% Penicillin / Streptomycin. 5-FdU Lager: 100x Lager, 1 mM 5-Fluor-2&#39;-desoxyuridin (Sigma F0503) and1mM Uridin (Sigma U3003) in Neurobasalmedium. Filter sterilisiert und gelagert als Aliquots bei -20 ° C. Papainverdau Lösung (make frisch vor Dissektion): <ol><li> Lösen 3,2 mg L-Cystein (Sigma C-7352) in 4ml DM </li><li> Der pH-Wert auf etwa 7,4 mit 1N NaOH (Test auf pH-Teststreifen) und im Wasserbad bei 37 ° C Hinweis: Führen Sie den nächsten Schritt nach rechts, bevor Gewebe Verdauung (siehe unten). </li><li> Add Papain, um eine endgültige Konzentration von 20 Einheiten / ml </li><li> Filter sterilisieren (0,22 mm), und in Wasserbad bei 37 ° C </li></ol> Trypsin-Inhibitor-Lösung (make frisch vor Dissektion): <ol><li> Lösen Sie 0,2 g Trypsin-Inhibitor (Sigma T7295) in 20ml DM </li><li> Überprüfen Sie pH und pH-Wert auf ~ 7,4 mit 1 N NaOH </li><li> Filter sterilisieren und in Wasserbad bei 37 ° C </li></ol> Brain-Dissection Setup: <ul><li> Sterilisiert Zangen und Scheren </li><li> 70% Ethanol </li><li> Saubere Seidenpapier und DIAPer Pad auf der Bank </li><li> 10cm Petrischalen mit eiskaltem DM für Gebärmutter und Fötus auf der Bank </li><li> Ice Plattform Dissektionsmikroskop </li><li> 2-3 6cm Gerichte mit DM auf Eis </li><li> Warm PM in einem 37 ° C Wasserbad </li></ul><ol><li> Euthanize eine schwangere Sprague-Dawley Ratten (E16) nach einem Verfahren von Ihrem Institutional Animal Care genehmigt und uns Committee (IACUC) </li><li> Legen Sie die Ratte auf die Windel-Pad und spay Alkohol auf die Bauchregion </li><li> Sie mit einer Pinzette auf die Bauchhaut und eine Schere zu halten, um eine V-Form Schnitt schneiden nur die Haut </li><li> Gespreizten der Haut und verwenden Sie ein anderes sterilisiert Pinzette und Schere, um durch die Muskelschicht geschnitten </li><li> Entpacken Sie die Kette der Embryosäcke und überführen sie in eine 10 cm Schüssel mit eiskaltem DM </li><li> Mit einer Mikrodissektion Schere, vorsichtig jedem Embryo aus dem Sack und das Gehirn von jedem Embryo, und legen Sie befreite Gehirn in eine saubere Schüssel mit 10cm DM auf ein Eis-Plattform </li><li> In einem Laminar Kapuze und unter einem Dissektionsmikroskop, entfernen Mittelhirn / Hinterhirn Abschnitte, und verwenden Sie feinen Pinzette Meningen zu entfernen, und übertragen Sie die gereinigt Kortex / Hippocampus, eine neue 6cm Schale mit DM auf einen Eisbeutel Hinweis: In dieser Phase wird das Gewebe bereit für die Enzym-Verdauung. </li><li> Machen Papain und Trypsin-Inhibitor-Lösung und Filter sterilisieren </li><li> Entfernen Sie überschüssiges DM aus Rinden / hippocampi </li><li> Add 4ml der vorbereiteten Papainverdau Lösung </li><li> Übertragen Sie alle Inhalte auf einen 50ml Tube und Ort der Falcon-Röhrchen in ein Wasserbad bei 37 ° C für genau 5 min </li><li> Entfernen Papain-Lösung mit Pipette </li><li> Add 5ml Trypsin-Inhibitor-Lösung, wirbeln die Röhre, und in einem Wasserbad bei 37 ° C für 2-3 min </li><li> Entfernen Trypsin-Inhibitor-Lösung </li><li> Wiederholen Sie Schritt 13 und 14 dreimal </li><li> Fügen Sie 20ml warmen PM </li><li> Verreiben, bis alle Klümpchen verschwunden sind (~ 20-30 mal) </li><li> Zentrifuge bei 100xg für 7min </li><li> Das Pellet in 10ml PM und waschen zweimal durch Zentrifugation </li><li> Das Pellet in 10ml PM für regelmäßige Neuron Kulturen oder in NBB27/DMEM für aggregierte Kulturen </li><li> Pass die Zellen durch ein 70μm-Zelle Siebe </li><li> Graf lebenden Zellen </li><li> Gehen Sie zu aggregieren Kulturen wie unten beschrieben oder Platte die Zellen für die regelmäßige Neuronenkulturen Hinweis: Bei diesem Schritt, hoch angereichertes Neuron Kulturen können durch Ausplattieren der dissoziierten Zellen auf PDL-Beschichtung Platten bei einer Dichte von 200-640 Zellen / mm 2 je nach Zweck der Experimente erreicht werden. Nachdem die Zellen gebunden haben (~ 2-4h), ersetzen Medium mit warmen Uhr. Wenn Kultivierung für längere Zeit, am Tag 4, ersetzen Sie die Hälfte des Mediums mit warmem CM. Für hochreine neuronalen Kulturen, mitotische Inhibitor 5-FdU (Endkonzentration 10 nM) sind DIV2 hinzugefügt werden, um nicht-neuronalen Zell-Proliferation (dh während DIV2-3) zu hemmen. Nach der Erholung in CM für 2 Tage werden die Zellen Puls wieder mit 5-FdU für weitere 2 Tage (DIV 6-7) durch eine vollständige Mediumwechsel behandelt, gefolgt. Danach ersetzen die Hälfte des Mediums mit frischem, warmen CM alle 3-4 Tage. Neuronen sind für bis zu 4 Wochen lebensfähig. </li></ol> Aggregates Vorbereitung und Beschichtung <ol><li> Suspend dissoziierten Zellen in NBB27/DMEM Medium mit 1x Sato, 10ng/ml CNTF und 10 um Forskolin bei einer Dichte von 2x10 6 Zellen / ml </li><li> Transfer-2ml Zellsuspension in jede Vertiefung einer unbeschichteten 6-Well-Platte </li><li> Kultur für 3 Nächte. Jeder Tag, sanft resuspendieren die Zellen einmal mit einem P1000 Pipetman Hinweis: Die Zellen beginnen zu Aggregaten (Abbildung 1). </li><li> Am Tag vor der gesamten Beschichtung, beschichten Platte / Deckgläser mit Matrigel: <ul><li> Verdünnen Lager Matrigel (growth factor reduziert, BD Biosciences 354230) 1:20 mit kaltem DMEM auf Eis Hinweis: Matrigel Aliquots sollten pro Hersteller-Protokoll vorbereitet werden. Alle Tipps, sollte Eppendorf-Röhrchen und Lösungen für die Herstellung Aliquots der Bestand sich kalt an Matrigel Polymerisation zu vermeiden. Aliquots von Matrigel werden bei -80 ° C gehalten und wieder aufgetaut bei 4 ° C. </li><li> Coat zuvor PDL-beschichtete Deckgläser mit 300μl/well der verdünnten Lösung Matrigel über Nacht in einem 37 ° C Inkubator </li><li> Mit warmem PBS, dann waschen Teller mit warmen sterilen ddH 2 O und lassen Sie die Platten in den Inkubator. </li></ul></li><li> 3 Tage nach der Aggregatbildung, sanft die Zellen wieder und Sieb die Suspension durch ein 200 um Maschenweite in ein 50ml Falcon-Röhrchen. Lassen Aggregate zum Boden des Röhrchens durch die Schwerkraft (~ 3-5min) begleichen </li><li> Entfernen Sie vorsichtig überstehende </li><li> Add Medium vorsichtig resuspendieren der Aggregate und lassen Sie sie wieder absetzen. Wiederholen Sie diesen Vorgang mehrmals, um abgestorbene Zellen, einzelne Zellen und Ablagerungen zu entfernen. Verwenden Mikroskop zu prüfen, ob der Überstand enthalten noch nicht aggregierten Zellen und Fremdkörpergefühls </li><li> Vorsichtig resuspendieren die Aggregate im gleichen NBB27/DMEM Medium (Abbildung 1) und zählen Aggregate </li><li> Passen Sie die Dichte der Aggregate zu etwa 25-30 aggreggats/50ul </li><li> Transfer-Aliquots (500-1000μl) des Aggregats Suspension 2ml Eppendorf-Röhrchen für die Beschichtung Hinweis: Da Aggregate zu beruhigen, um den Boden der Rohre neigen, ist es wichtig, mehrere Rohre des Aggregats Aufhängung für die Beschichtung zu machen. Vorsichtig resuspendieren die Aggregate häufig vor der Aussaat sie auf beschichtete Deckgläser oder Kultur Platten ähnliche Anzahl von Aggregaten auf jeder Deckglas vernickelt gewährleisten. </li><li> Nehmen Sie die Kultur Platte mit Matrigel-beschichteten Deckgläser Inkubator. Remove-Lösung aus jeder Vertiefung, waschen Sie die Deckgläser mit warmem PBS und ddH 2 O, und dann kurz antrocknen. Sie sind nun für insgesamt Aussaat bereit </li><li> Invert ein Röhrchen mit insgesamt Aufhängung, damit sie gleichmäßig vor der Aussaat verteilt werden. Legen Sie 50 ul der aggregierten Suspension in die Mitte jedes Deckglas, und legte sanft die Platte zurück zum Inkubator ohne weitere Störung zu ermöglichen Aggregate gleichmäßig Wert auf das Deckglas Hinweis: Die Dichte der Aggregate ist von entscheidender Bedeutung. Wenn Aggregate ausgesät sind zu nahe beieinander oder wenn die Aussaatdichte hoch ist, neigen sie dazu, miteinander verschmelzen, wie sie wachsen. </li><li> Fügen Sie zusätzliche DMEM/NBB27medium (500 ul) in jede Vertiefung 4-6 h später oder am nächsten Morgen früh </li><li> Um die Aggregat-Kultur, die Hälfte ändern das Medium alle 3-4 Tage. Wenn Veränderungen mittel, darauf achten, nicht zu den Aggregaten zu stören und dies wird durch Zugabe von Medium sanft entlang der Seitenwand des gut gemacht. Hinweis: Nur wenige Stunden nach der Aggregate an die Platte, Neuritenwachstum von der Aggregate beobachtet werden können. Axone wachsen schnell in den ersten zwei Wochen und bilden reichlich Verbindungen zwischen den Aggregaten (Abbildung 2). Gliazellen Vorfahren radial wandern aus der Aggregate und differenzieren im Laufe der Zeit in Astrozyten und Oligodendrozyten reifen. </li></ol><img src="/files/ftp_upload/1304/1304fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Phase kontrastreiche Bilder von Zellen bilden Aggregate an verschiedenen Tagen und Aggregate nach dem Waschen. <img src="/files/ftp_upload/1304/1304fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Phase kontrastreiche Bilder eines Aggregats Kultur 2 und 12 Tage nach ausgesät auf Matrigel-beschichteten Deckgläschen.

<ol>
	<li>Matthieu, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelination+in+rat+brain+aggregating+cell+cultures.">Myelination in rat brain aggregating cell cultures.</a> Neuroscience. 3, 565-56 (1978).</li><li>Chen, Y. i. n. g. <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+culture+of+rat+and+mouse+oligodendrocyte+precursor+cells.">Isolation and culture of rat and mouse oligodendrocyte precursor cells.</a> Nat. Protocols. 2, 1044-10 (2007).</li><li>Watkins, T. A., Emery, B., Mulinyawe, S., 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=Distinct+stages+of+myelination+regulated+by+gamma-secretase+and+astrocytes+in+a+rapidly+myelinating+CNS+coculture+system.">Distinct stages of myelination regulated by gamma-secretase and astrocytes in a rapidly myelinating CNS coculture system.</a> Neuron. 60, 555-55 (2008).</li><li>Svenningsen, A. F., Shan, W. e. i. -. S. o. n. g., Colman, D. a. v. i. d. R., Pedraza, L. i. l. i. a. n. a. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+method+for+culturing+embryonic+neuron-glial+cell+cocultures.">Rapid method for culturing embryonic neuron-glial cell cocultures.</a> Journal of Neuroscience Research. 72, 565-56 (2003).</li></ol>

Frühe Studien berichteten Bildung von Synapsen und reife Myelin in Rotation vermittelte frei schwebenden aggregierten Kulturen 1. Das ZNS Aggregat-Kultur hier beschriebene System vereint serumfreies Wachstum von multipotenten Vorläuferzellen in dreidimensionalen Aggregaten mit dem Komfort der traditionellen 2D-Kulturen auf Analysen der Zell-Entwicklung, Migration und Differenzierung in vitro zu erleichtern. Das System kann modifiziert und für neurale Vorläuferzellen verwendet werden Stam...

preparation of rat brain aggregate cultures for neuron and glia development studies

Eine In-vitro-System, rekapituliert die Entwicklung und Differenzierung von Vorläuferzellen zu reifen Neuronen und Gliazellen im zentralen Nervensystem (ZNS), wäre eine leistungsstarke Plattform für Neurowissenschaftler liefern zu axo-gliale Interaktionen, Eigenschaften und Differenzierung von multipotenten Vorläuferzellen, und das Fortschreiten der oligodendroglialen untersuchen Linie Zellen auf zellulärer und molekularer Ebene. Wir beschreiben hier eine ZNS-Aggregat-Kultur-System aus embryonalen Ratte Vorderhirn, die in einem Serum-freiem Medium gehalten werden können bis zu 3-4 Wochen und ist in unserem Labor als Modell zu Neuron-Glia-Interaktion und ZNS Myelinisierung Studie verwendet. Dieses Video wird gezeigt, wie zu isolieren und zu wachsen diese CNS aggregierten Kulturen aus E16 Rattenhirn. Ferner ist aus dem gleichen Gehirn Dissektion kann hochangereichertes regelmäßigen dissoziierten neuronalen Kulturen leicht erhalten werden und für verschiedene Studien über ZNS-Neuronen oder für Co-Kulturen mit anderen Zellen.

Watch this Scientific Journal Video about Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies at JoVE.com

Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies

A-Protokolle für eine embryonale Rattenhirn Aggregat-Kultur-System beschrieben wird. Multipotente Vorläuferzellen in die Aggregate entwickeln können und differenzieren sich in Neuronen, Astrozyten und Oligodendrozyten.

Herstellung von Ratten-Gehirn-Aggregate Kulturen für Neuron und Glia Development Studies

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) ...

preparation-rat-brain-aggregate-cultures-for-neuron-glia-development

Research

JoVE Journal

Biology

12.3K Aufrufe.  Texas A&M University (TAMU). A-Protokolle für eine embryonale Rattenhirn Aggregat-Kultur-System beschrieben wird. Multipotente Vorläuferzellen in die Aggregate entwickeln können und differenzieren sich in Neuronen, Astrozyten und Oligodendrozyten.

Serie: Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.

Primäre neuronale Kulturen aus den Gehirnen von Late Stage Drosophila Puppen

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

Forschung

Biologie

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

Vorbereitung der neuronalen Kulturen aus Midgastrula Bühne Drosophila Embryonen

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

ALS - Motor Neuron Disease: Mechanism and Development of New Therapies

Jeffrey D. Rothstein speaks about the pathology and mechanisms underlying amyotrophic lateral sclerosis or ALS, advances in ALS research, and current strategies towards the development of therapies.

ALS - Motor Neuron Disease: Mechanismus und Entwicklung neuer Therapien

Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for culturing neurons. Previously, a silicon wafer was patterned with the design for the neuron microfluidic device using SU-8 and photolithography to create a master mold, or what we simply refer to as a "master". Next, we pour the silicon polymer PDMS on top of the master which is then cured by heating the PDMS to 80&deg;C for 1 hour. The PDMS forms a negative mold of the device. The PDMS is then carefully cut and lifted away from the master. Holes are punched where the reservoirs will be and the excess PDMS trimmed away from the device. Nitrogen is used to blow away any excess debris from the device. At this point the devices are now ready for use and can either bonded to corning No. 1 cover glass with a plasma sterilizer/cleaner or can be reversibly bound to the cover glass by simply placing the device on top of the cover glass. The reversible bonding of the device to glass is covered in a separate video and requires first that the device be sterilized either with 70% ethanol or by autoclaving. Plasma treating sterilizes the devices so no further treatment is necessary. It is, however, important, when plasma-treating the devices, to add liquid to the devices within 10 minutes of the plasma treatment while the surfaces are still hydrophilic. Waiting longer than 10 minutes to add liquid to the device makes it difficult for the liquid to enter the device. The neuron devices are typically plasma-bound to cover glass and 0.5 mg/ml poly-L-lysine (PLL) in pH 8.5 borate buffer is immediately added to the device. After a minimum of 3 hours incubating with PLL, the devices are washed with dH2O water a minimum of 3 times with at least 15 minutes between each wash. Next, the water is removed and fresh media is added to the device. At this point the device is ready for use. It is important to remember at this point to never remove all the media from the device. Always leave media in the main channel.

In this video we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to farbricate a microfluidic device for culturing neurons.

Herstellung eines mikrofluidischen Vorrichtung für die Abschottung der Neuron Soma und Axonen

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for ...

Preparation of Dissociated Mouse Cortical Neuron Cultures

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures can be used for a variety of applications including immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging, protein and/or RNA isolation. These cultures also provide a platform to study the neuronal development of transgenic animals that carry a late embryonic or postnatal lethal gene mutation. The procedure is relatively straight forward, requires some experience in tissue culture technique and should not take longer than two to three hours if you are properly prepared. Careful separation of the cortical rind from the thalamo-cortical fiber tract will reduce the number of unwanted non-neuronal cells. To increase yields of neuronal cells triturate the pieces of the cortical tissue gently after the enzyme incubation step. This is imperative as it prevents unnecessary injury to cells and premature neuronal cell death. Since these cultures are maintained in the absence of glia feeder cells, they also offer an added advantage of growing cultures enriched in neurons.

This video shows a procedure for generating neuronal cultures from late embryo and early postnatal mouse cortex. These cultures can be used for immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging and provide a platform to study the neuronal development of transgenic animals that carry a postnatal lethal gene mutation.

Vorbereitung von dissoziierten Maus kortikale Neuron Kulturen

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures ...

Preparation of 2-dGuo-Treated Thymus Organ Cultures

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play a key role in the development of a functionally competent T-cell pool. However, the complexity of T-cell development in the thymus in vivo can limit analysis of individual cellular components and particular stages of development. In vitro culture systems provide a readily accessible means to study multiple complex cellular processes. Thymus organ culture systems represent a widely used approach to study intrathymic development of T-cells under defined conditions in vitro. Here we describe a system in which mouse embryonic thymus lobes can be depleted of endogenous haemopoeitic elements by prior organ culture in 2-deoxyguanosine, a compound that is selectively toxic to haemopoeitic cells. As well as providing a readily accessible source of thymic stromal cells to investigate the role of thymic microenvironments in the development and selection of T-cells, this technique also underpins further experimental approaches that include the reconstitution of alymphoid thymus lobes in vitro with defined haemopoietic elements, the transplantation of alymphoid thymuses into recipient mice, and the formation of reaggregate thymus organ cultures. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).

This video demonstrates the dissection and removal of the fetal thymus as well the preparation of ex vivo cultures of 2-dGuo-treated thymus.

Herstellung von 2-dGuo-Behandelte Thymus Organkulturen

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play ...

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons. The neurons are large (up to 1 mm in diameter) and identifiable, with distinct sizes, shapes, positions and pigmentations, and the cell bodies are externally exposed in five paired ganglia distributed throughout the body of the animal. These properties have allowed investigators to delineate the circuitry underlying specific behaviors in the animal1. The monosynaptic connection between sensory and motor neurons is a central component of the gill-withdrawal reflex in the animal, a simple defensive reflex in which the animal withdraws its gill in response to tactile stimulation of the siphon. This reflex undergoes forms of non-associative and associative learning, including sensitization, habituation and classical conditioning. Of particular benefit to the study of synaptic plasticity, the sensory-motor synapse can be reconstituted in culture, where well-characterized stimuli elicit forms of plasticity that have direct correlates in the behavior of the animal2,3. Specifically, application of serotonin produces a synaptic strengthening that, depending on the application protocol, lasts for minutes (short-term facilitation), hours (intermediate-term facilitation) or days (long-term facilitation). In contrast, application of the peptide transmitter FMRFamide produces a synaptic weakening or depression that, depending on the application protocol, can last from minutes to days (long-term depression). The large size of the neurons allows for repeated sharp electrode recording of synaptic strength over periods of days together with microinjection of expression vectors, siRNAs and other compounds to target specific signaling cascades and molecules and thereby identify the molecular and cell biological steps that underlie the changes in synaptic efficacy.
An additional advantage of the Aplysia culture system comes from the fact that the neurons demonstrate synapse-specificity in culture4,5. Thus, sensory neurons do not form synapses with themselves (autapses) or with other sensory neurons, nor do they form synapses with non-target identified motor neurons in culture. The varicosities, sites of synaptic contact between sensory and motor neurons, are large enough (2-7 microns in diameter) to allow synapse formation (as well as changes in synaptic morphology) with target motor neurons to be studied at the light microscopic level.
In this video, we demonstrate each step of preparing sensory-motor neuron cultures, including anesthetizing adult and juvenile Aplysia, dissecting their ganglia, protease digestion of the ganglia, removal of the connective tissue by microdissection, identification of both sensory and motor neurons and removal of each cell type by microdissection, plating of the motor neuron, addition of the sensory neuron and manipulation of the sensory neurite to form contact with the cultured motor neuron.

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

Primary cultures of Aplysia sensory-motor neurons provide a model preparation for studying synapse formation and synaptic plasticity in vitro. This video demonstrates the identification and microdissection of sensory and motor neurons from Aplysia ganglia as well as the methods for establishing and maintaining sensory-motor neurons in culture.

Vorbereitung der Aplysia Sensory-Motor neuronalen Zellkulturen

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons.  The neurons are large (up ...

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability.
The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput.
Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 &mu;m) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master.
Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

Ein Multi-Kompartiment-ZNS-Neuron-Glia-Co-Kultur mikrofluidischen Plattform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to ...

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, moniliformin, etc... have adverse effects on both human and animal health and some are considered as pathogenicity factors. Proteomic studies showed to be effective for deciphering toxin production mechanisms (Taylor et al., 2008) as well as for identifying potential pathogenic factors (Paper et al., 2007, Houterman et al., 2007) in Fusaria. It becomes therefore fundamental to establish reliable methods for comparing between proteomic studies in order to rely on true differences found in protein expression among experiments, strains and laboratories. The procedure that will be described should contribute to an increased level of standardization of proteomic procedures by two ways. The filmed protocol is used to increase the level of details that can be described precisely. Moreover, the availability of standardized procedures to process biological replicates should guarantee a higher robustness of data, taking into account also the human factor within the technical reproducibility of the extraction procedure.
The protocol described requires 16 days for its completion: fourteen days for cultures and two days for protein extraction (figure 1). 
Briefly, Fusarium strains are grown on solid media for 4 days; they are then manually fragmented and transferred into a modified toxin inducing media (Jiao et al., 2008) for 10 days. Mycelium is collected by filtration through a Miracloth layer. Grinding is performed in a cold chamber. Different operators performed extraction replicates (n=3) in order to take into account the bias due to technical variations (figure 2). Extraction was based on a SDS/DTT buffer as described in Taylor et al. (2008) with slight modifications. Total protein extraction required a precipitation process of the proteins using Aceton/TCA/DTT buffer overnight and Acetone /DTT washing (figure 3a,3b). Proteins were finally resolubilized in the protein-labelling buffer and quantified. Results of the extraction were visualized on a 1D gel (Figure 4, SDS-PAGE), before proceeding to 2D gels (IEF/SDS-PAGE). The same procedure can be applied for proteomic analyses on other growing media and other filamentous fungi (Miles et al., 2007).

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Protein extraction for proteomic analyses in fungal species requires high levels of standardization to be accomplished according with the minimum information about a proteomic experiment (MIAPE) guidelines. We present a video-protocol that includes a procedure for minimizing experimental bias during toxin induction and protein extraction from Fusarium spp.

Toxin Induktions-und Protein-Extraktion aus Fusarium Spp. Kulturen für Proteom-Studien

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, ...

Preparation of Rat Tail Tendons for Biomechanical and Mechanobiological Studies

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working with those tissues is challenging because they are very fragile, and until now there was no rigorously detailed protocol for their isolation.
Faced with these challenges, we have developed methods and instruments to facilitate manipulation of RTTs and control tissue viability, sterility and integrity. This article describes the experimental procedures used to prepare RTTs for biomechanical and mechanobiological studies. Our work is divided into four main steps: extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.
At each step, all procedures, materials and manipulations are presented in detail so that they can be easily reproduced. Moreover, the specific instruments developed are presented: a manipulation plate used to segregate RTTs, an optic micrometer to position the tissue during the cross-sectional area measurement and an anchoring system to attach the RTTs onto a bioreactor.
Finally, we describe the results obtained after multiple tests to validate our methods. The viability, sterility and integrity evaluations demonstrate that our procedures are sufficiently rigorous for manipulations of fragile tissues such as rat tail tendons.

This article describes the experimental procedures used to prepare rat tail tendons for biomechanical and mechanobiological studies. Several features of the main steps in preparation are demonstrated, beginning with extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.

Vorbereitung der Rattenschwanzsehnen für Biomechanik und Mechanobiological Studies

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working ...