Cette étude a été financée en partie par des fonds de démarrage de la Texas A &amp; M University

Préparation avant dissection Outils chirurgicaux: stériliser tous les ciseaux de dissection et une pince en autoclave Nettoyage de la lamelle: <ol><li> Faire tremper les lamelles (15mm de diamètre pour 24 puits, plaque) dans 1L bécher herbe dans 33% de HCl pendant au moins 24 h </li><li> Laver à l&#39;eau courante pendant 10 min avec secouez occasionnelle pour supprimer tous les résidus de HCl </li><li> Rincer à l&#39;eau Millipore </li><li> Égoutter l&#39;eau hors </li><li> Faire tremper dans de l&#39;éthanol lamelles 95-98% </li><li> Lamelles Transférer dans un mouchoir en papier propre sur une plaque plate et l&#39;air sec les lamelles </li><li> Transfert des lamelles d&#39;un récipient en verre, couvrir de papier aluminium et autoclaver Remarque: Lamelles peuvent être stockées à ce stade </li><li> Placer la lamelle individuels dans des boîtes de culture. Prêt pour le revêtement </li></ol> Lamelle de revêtement: <ol><li> Diluer 100 x stocks de poly-D-lysine (PDL, 10 mg / ml dans du sérum bovin 0,5% d&#39;albumine dans le PBS, stockées sous forme d&#39;aliquots à -20 ° C) avec du PBS et le filtre de stériliser (0,22 um) </li><li> Manteau lamelles avec 1 x PDL solution (~ 0,15 ml / cm 2) pendant 2 h à 37 ° C dans un incubateur </li><li> Retirer solution de PDL et laver 3 fois avec O stériles ddH 2 et lamelles sécher complètement </li></ol> Remarque: Le poil assez de lamelles / plaques pour chaque dissection. PDL-revêtement des lamelles peuvent être stockées pendant quelques semaines à 4 ° C. Médias et des solutions Dissection moyenne (DM): Utiliser une solution stérile glacée saline équilibrée de Hank (w / o Ca 2 +, Mg 2 +) (HBSS, Invitrogen 14175) supplémenté avec 10 mM HEPES (Invitrogen 15630). Milieux de culture globale: Le milieu contient DMEM/NBB27 DMEM / Neurobasal (1:1 vol: vol), 2% B27, 1 x Sato, 0,5 mM pyruvate de sodium, 0,75 glutamax, 60μg/ml N-acétylcystéine, 5 pg / ml insuline, 10 nM d-biotine et 1% de pénicilline / streptomycine. Pour faire 100 ml de milieu de culture globale, mélanger 50 ml de DMEM (w / o pyruvate / glutamine, Invitrogen 11960), 50 ml de milieu Neurobasal (Invitrogen 211 034), 375 ul glutamax (100 x 35 050 Invitrogen), 5,5 mg de sodium pyruvate (Sigma p2256), 2 ml B27 (Invitrogen 17504), 6,3 mg N-acétylcystéine (Sigma A8199), 1 ml de Sato des stocks (100 x stocks, voir ci-dessous), 25 pl de d-biotine stock (4000 x stock, 40 uM dans du PBS stockées sous forme d&#39;aliquots à -20 ° C. Sigma B4639), 100 ul de l&#39;insuline (1000 x stock, 5 mg / ml dans HCl 0,01 N stockées sous forme d&#39;aliquots à -20 ° C, Sigma 16 634), et 1 ml de pénicilline / streptomycine (100 x stock, Invitrogen 15140), filtre à stériliser et stocker à 4 ° C. Sato 100 x solution stock: Pour 40 ml du stock: mélanger 40 ml Neurobasal avec 400 mg d&#39;apo-transferrine (Sigma T2252), 400 mg de BSA (Sigma A9647), 10 pl de la progestérone (25 mg / ml d&#39;éthanol, stockées sous forme de aliquotes à -20 ° C. Sigma P8783), 64 mg putrescine (Sigma P7505), et 40 pi de sélénite de sodium (30 uM dans du PBS, stockées sous forme d&#39;aliquots à -20 ° C, Sigma S5261). Filtre stériliser la solution stock de Sato, et de stocker en aliquotes à -20 ° C. Milieu d&#39;étalement Neuron (PM): Moyen Neurobasal, 2% B27, 2 mM glutamine (stock 100x, aliquotes conservés à -20 ° C, Invitrogen 25030), 25 um d&#39;acide glutamique (100 stock, aliquotes conservés à -20 ° C) et 1% pénicilline / streptomycine. Milieu de culture Neuron (CM): Moyen Neurobasal, 2% B27, 2 mM glutamine (stock 100x, aliquotes conservés à -20 ° C) et 1% de pénicilline / streptomycine. 5 FdU Stock: 100x, 1 mM de 5-fluoro-2&#39;-désoxyuridine (Sigma F0503) and1mM uridine (Sigma U3003) en milieu Neurobasal. Filtre stérilisés et stockés sous forme d&#39;aliquots à -20 ° C. Solution de digestion par la papaïne (assurez frais avant dissection): <ol><li> Dissoudre 3,2 mg de L-cystéine (Sigma C-7352) dans 4ml DM </li><li> Ajuster le pH à environ 7,4 avec NaOH 1N (test sur bandelettes pH) et placer dans un bain d&#39;eau à 37 ° C Remarque: Effectuez l&#39;étape suivante juste avant la digestion des tissus (voir ci-dessous). </li><li> Ajouter la papaïne à une concentration finale de 20 unités / ml </li><li> Stériliser par filtration (0,22 mm), et placer dans un bain d&#39;eau à 37 ° C </li></ol> Solution d&#39;inhibiteur de trypsine (assurez frais avant dissection): <ol><li> Dissoudre 0,2 g d&#39;inhibiteur de trypsine (Sigma T7295) dans 20ml DM </li><li> Vérifier le pH et ajuster le pH à ~ 7,4 avec NaOH 1 N </li><li> Stériliser par filtration, et placer dans un bain d&#39;eau à 37 ° C </li></ol> Dissection du cerveau Configuration: <ul><li> Pinces stérilisées et ciseaux </li><li> 70% d&#39;éthanol </li><li> Papiers tissu propre et DIAPER pad sur le banc </li><li> Boîtes de Pétri de 10 cm avec glacée DM pour l&#39;utérus et le fœtus sur le banc </li><li> Plate-forme de glace au microscope à dissection </li><li> 2-3 plats 6cm avec DM sur la glace </li><li> PM chaud dans un bain à 37 ° C l&#39;eau </li></ul><ol><li> Euthanasier un rates Sprague-Dawley rats (E16) selon une procédure approuvée par votre Animal Care institutionnels et nous Comité (IACUC) </li><li> Posez le rat sur le pavé de couches et de l&#39;alcool spay sur la région abdominale </li><li> Utilisez une pince à épiler pour maintenir la peau abdominale et une paire de ciseaux pour faire une incision en forme de V uniquement la coupe de la peau </li><li> Écartez la peau et utiliser un autre pincettes stérilisées et les ciseaux pour couper à travers la couche musculaire </li><li> Extrait de la chaîne des sacs embryon et les transférer dans un plat de 10cm contenant glacée DM </li><li> En utilisant une paire de ciseaux de microdissection, retirez soigneusement chaque embryon du sac et le cerveau de chaque embryon, et la place libérée des cerveaux dans un plat de 10cm nettoyer avec DM sur une plate-forme de glace </li><li> Dans une hotte laminaire et sous un microscope de dissection, retirer du mésencéphale / rhombencéphale sections, et d&#39;utiliser une pince fine pour retirer les méninges, et le transfert du cortex nettoyé / hippocampe d&#39;un nouveau plat 6cm avec DM sur un sac de glace Note: à ce stade, le tissu est prêt pour la digestion enzymatique. </li><li> Faire la papaïne et la solution d&#39;inhibiteur de trypsine et stériliser par filtration </li><li> Enlever l&#39;excès de DM cortex / hippocampes </li><li> Ajouter 4 ml de la solution préparée digestion par la papaïne </li><li> Transfert tout le contenu d&#39;un tube de 50 ml et placer le tube Falcon dans un bain d&#39;eau à 37 ° C pendant exactement 5 minutes </li><li> Retirer solution de papaïne avec pipette </li><li> Ajouter 5 ml solution inhibiteur de la trypsine, agiter le tube, et les placer dans un bain d&#39;eau à 37 ° C pendant 2-3 min </li><li> Retirer solution inhibiteur de la trypsine </li><li> Répétez l&#39;étape 13 et 14 trois fois </li><li> Ajouter 20ml PM chaude </li><li> Triturer jusqu&#39;à ce que tous touffes ont disparu (~ 20-30 fois) </li><li> Centrifuger à 100xg pour 7min </li><li> Reprendre le culot dans 10ml PM et laver deux fois par centrifugation </li><li> Reprendre le culot dans 10ml PM pour des cultures de neurones régulière ou dans NBB27/DMEM pour les cultures globales </li><li> Passez les cellules à travers un tamis cellulaire 70μm </li><li> Nombre des cellules vivantes </li><li> Procéder à des cultures globales décrites ci-dessous ou la plaque des cellules pour des cultures de neurones régulière Note: À cette étape, hautement enrichi des cultures de neurones peut être atteint par les cellules dissociées plaquage sur PDL-revêtement des plaques à une densité de 200 à 640 cellules / mm 2 selon l&#39;objet d&#39;expérimentations. Après les cellules ont attaché (~ 2-4h), remplacer moyenne avec PM chaud. Si la culture pendant de longues périodes de temps, le jour 4, remplacer la moitié de la moyenne avec CM chaud. Pour hautement purifiée cultures de neurones, inhibiteur mitotique 5-UPF (concentration finale 10 pM) peuvent être ajoutés à DIV2 pour inhiber la prolifération des cellules non-neuronales (c&#39;est à dire, pendant DIV2-3). Après la récupération de CM pendant 2 jours, les cellules sont traitées à nouveau d&#39;impulsion avec le 5-FdU pendant 2 jours (DIV 6-7) suivie d&#39;un changement de milieu complet. Par la suite, remplacez la moitié de la moyenne avec des produits frais, CM chaude tous les 3-4 jours. Les neurones sont viables jusqu&#39;à 4 semaines. </li></ol> Préparation de granulats et de placage <ol><li> Suspendre les cellules dissociées en milieu NBB27/DMEM contenant 1x Sato, 10ng/ml CNTF et 10 uM de forskoline à une densité de 2x10 6 cellules / ml </li><li> Transfert 2ml de suspension cellulaire dans chaque puits d&#39;une non couchés à 6 puits plaque </li><li> Culture pour 3 nuits. Chaque jour, doucement remettre en suspension les cellules une fois en utilisant un Pipetman P1000 Remarque: les cellules commencent à former des agrégats (Figure 1). </li><li> Le jour avant l&#39;étalement global, manteau plaque / lamelles de Matrigel: <ul><li> Diluer stock de Matrigel (facteur de croissance réduite, BD Biosciences 354230) 01:20 avec le froid DMEM sur la glace Remarque: aliquotes Matrigel devrait être préparé par le protocole du fabricant. Toutes les astuces, des tubes Eppendorf et des solutions pour faire des aliquotes du stock doit être froide pour éviter la polymérisation Matrigel. Des aliquotes de Matrigel sont conservés à -80 ° C et décongelés à 4 ° C. </li><li> Manteau préalablement enduit de PDL lamelles avec 300μl/well de la solution diluée Matrigel nuit dans un incubateur à 37 ° C </li><li> Laver avec du PBS tiède, puis lavez-plaque avec chaleureuse stériles O ddH 2 et laissez les plaques dans l&#39;incubateur. </li></ul></li><li> 3 jours après la formation d&#39;agrégats, doucement resuspendre les cellules à nouveau et tamisez la suspension à travers un maillage de 200μm dans un tube de 50ml Falcon. Autoriser les agrégats se déposent au fond du tube par gravité (~ 3-5min) </li><li> Retirer délicatement le surnageant </li><li> Ajouter moyen, délicatement remettre en suspension les agrégats et les laisser s&#39;installer de nouveau. Répétez cette procédure plusieurs fois pour éliminer les cellules mortes, les cellules individuelles et de débris. Utilisez microscope pour vérifier si le surnageant contiennent encore non agrégées cellules et debris </li><li> Resuspendre doucement les agrégats dans la moyenne NBB27/DMEM mêmes (figure 1) et le nombre d&#39;agrégats </li><li> Réglez la densité d&#39;agrégats d&#39;environ 25-30 aggreggats/50ul </li><li> Aliquotes de transfert (500-1000μl) de la suspension totale de 2ml tubes Eppendorf pour le placage Remarque: Comme les agrégats ont tendance à s&#39;installer au fond des tubes, il est crucial de faire de multiples tubes de la suspension totale pour le placage. Resuspendre doucement les agrégats souvent avant les ensemençant sur des lamelles enduites ou des plaques de culture afin d&#39;assurer le même nombre d&#39;agrégats plaqués sur chaque lamelle. </li><li> Sortez la plaque de culture contenant de Matrigel enduit de lamelles de l&#39;incubateur. Retirer solution à partir de chaque puits, se laver les lamelles avec du PBS et chaleureuse ddH 2 O, puis brièvement à sec. Ils sont maintenant prêts pour l&#39;ensemencement global </li><li> Inverser un tube contenant la suspension totale de leur permettre d&#39;être distribué uniformément avant l&#39;ensemencement. Charge 50 pl de la suspension totale dans le centre de chaque lamelle, et posa doucement la plaque arrière de l&#39;incubateur sans aucune perturbation supplémentaire pour permettre d&#39;attacher des agrégats uniformément sur la lamelle Note: La densité des agrégats est cruciale. Si les agrégats sont ensemencées trop près les uns aux autres ou si la densité de semis est élevée, ils ont tendance à fusionner à mesure qu&#39;ils grandissent. </li><li> Ajouter DMEM/NBB27medium supplémentaires (500 pi) dans chaque puits 4-6 h plus tard ou tôt le lendemain matin </li><li> Afin de maintenir la culture globale, la moitié changer le milieu tous les 3-4 jours. Quand le changement moyen, attention à ne pas perturber les agrégats et cela se fait en ajoutant doucement moyenne le long du mur côté du bien. Remarque: Quelques heures après agrégats attaché à la plaque, la croissance des neurites des agrégats peuvent être observés. Les axones se développent rapidement dans les deux premières semaines et de former des connexions entre les agrégats abondants (figure 2). Progéniteurs gliales migrent radialement des agrégats et de se différencier au cours du temps en astrocytes et des oligodendrocytes matures. </li></ol><img src="/files/ftp_upload/1304/1304fig1.jpg" alt="Figure 1" /> Figure 1. Images par contraste de phase de cellules formant des agrégats à des jours différents et des agrégats après le lavage. <img src="/files/ftp_upload/1304/1304fig2.jpg" alt="Figure 2" /> Figure 2. Images par contraste de phase d&#39;une culture globale de 2 à 12 jours après ensemencées sur Matrigel revêtement des lamelles.

<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>

Les premières études a signalé la formation de synapses et de myéline mature dans la rotation des cultures libres à médiation globale flottant 1. Le système CNS culture globale décrite ici combine sans sérum de croissance des cellules progénitrices multipotentes en trois dimensions des agrégats avec la commodité de cultures traditionnelles en 2D pour faciliter les analyses du développement cellulaire, la migration et la différenciation in vitro. Le système peut être modifié et utilis...

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

Un système in vitro qui récapitule le développement et la différenciation des progéniteurs en neurones matures et des cellules gliales dans le système nerveux central (SNC) offrirait une plateforme puissante pour les neuroscientifiques pour enquêter sur les interactions axo-gliales, les propriétés et la différenciation des progéniteurs multipotentes, et la progression de oligodendrogliales cellules lignée au niveau cellulaire et moléculaire. Nous décrivons ici un système de culture de SNC agrégées de forebrains rats embryonnaires, qui peuvent être maintenues dans un milieu sans sérum à 3-4 semaines et est utilisé dans notre laboratoire comme un modèle pour étudier l&#39;interaction neurone-glie et la myélinisation du SNC. Ce clip vidéo vous montrera comment isoler et cultiver ces cultures SNC global du cerveau de rat E16. Par ailleurs, à partir de la dissection du cerveau même, hautement enrichi régulièrement dissociée cultures neuronales peuvent être facilement obtenues et utilisées pour les différentes études sur les neurones du SNC ou utilisés pour des co-cultures avec d&#39;autres cellules.

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

Un des protocoles pour un système de cerveau de rat culture embryonnaire agrégat est décrite. Progéniteurs multipotentes dans les agrégats peuvent se développer et se différencier en neurones, astrocytes et des oligodendrocytes.

Préparation des cultures de cerveau de rat d&#39;agrégation pour Neuron et études sur le développement Glia

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

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12.3K vues.  Texas A&M University (TAMU). Un des protocoles pour un système de cerveau de rat culture embryonnaire agrégat est décrite. Progéniteurs multipotentes dans les agrégats peuvent se développer et se différencier en neurones, astrocytes et des oligodendrocytes.

Vidéo: 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.

Cultures neuronales primaires du cerveau des drosophiles stade tardif pupes

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

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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.

Préparation des cultures de neurones d&#39;embryons de drosophile Midgastrula scène

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.

SLA - maladie du motoneurone: Mécanisme et développement de nouvelles thérapies

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.

Fabrication d&#39;un dispositif microfluidique pour la compartimentation du Neuron Soma et des axones

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.

Préparation de cultures souris Dissocié neurones corticaux

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.

Préparation de la 2-dGuo cultures traitées orgue Thymus

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.

Préparation des Aplysie Sensori-motrice cultures de cellules neuronales

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.

Un compartiment Multi-SNC neurone-glie co-culture de la plate-forme microfluidique

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.

L&#39;induction ou à toxines et d&#39;extraction de protéines à partir Fusarium Spp. Cultures pour les études protéomiques

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.

Préparation des tendons de queue de rat pour les études biomécaniques et mécanobiologique

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