Este estudo foi financiado em parte por fundos start-up da Universidade Texas A &amp; M

Preparação antes da dissecação Instrumentos cirúrgicos: esterilizar todas as tesouras e pinças de dissecção por autoclave Lamela de limpeza: <ol><li> Mergulhe todos lamínulas (15mm de diâmetro por 24 bem-placa) em 1L copo de grama em 33% HCl durante pelo menos 24 h </li><li> Lavar com água corrente durante 10 min com agitação ocasional para remover todos os HCl residual </li><li> Enxágüe com água Millipore </li><li> Drenar a água fora </li><li> Mergulhe lamínulas em etanol 95-98% </li><li> Lamínulas transferência para um tecido limpo em uma placa plana e ar seco as lamelas </li><li> Transferência de lamínulas para um copo de vidro, cubra com papel alumínio e autoclave Nota: Lamelas podem ser armazenados nesta fase </li><li> Lugar lamela individual em placas de cultura. Pronto para revestimento </li></ol> Lamela revestimento: <ol><li> Diluir 100 x estoque de poli-D-lisina (PDL, 10 mg / ml em 0,5% de soro albumina bovina em PBS, armazenado como alíquotas a -20 ° C) com PBS e filtro-esterilize (0,22 mm) </li><li> Lamínulas revestimento com uma solução PDL x (~ 0,15 ml / cm 2) por 2 horas a 37 ° C em uma incubadora </li><li> Remoção da solução PDL e lavar 3 vezes com estéril DDH 2 O e lamínulas secar completamente </li></ol> Nota: Brasão suficiente lamínulas / placas para cada dissecção. PDL revestido lamelas podem ser armazenadas por algumas semanas a 4 ° C. Mídia e soluções Dissecção médio (DM): Use soro fisiológico gelado de Hank salina balanceada (w / o Ca 2 +, Mg 2 +) (HBSS, Invitrogen 14175) suplementado com 10 mM HEPES (Invitrogen 15.630). Agregada meios de cultura: O meio DMEM/NBB27 contém DMEM / Neurobasal (1:1 vol: vol), 2% B27, 1 x Sato, 0,5 mM piruvato de sódio, 0,75 mm Glutamax, 60μg/ml N-acetilcisteína, 5 mg / ml insulina, 10nM d-Biotina e 1% de penicilina / estreptomicina. Para fazer 100 ml do meio de cultura agregada, misture 50 ml DMEM (w / o piruvato / glutamina, Invitrogen 11.960), 50 ml de meio de Neurobasal (Invitrogen 211.034), 375 Glutamax mL (100 x, Invitrogen 35.050), 5,5 mg de sódio piruvato (Sigma P2256), 2 ml B27 (17504 Invitrogen), 6,3 mg N-acetilcisteína (Sigma A8199), 1 ml de Sato ações (stock x 100, veja abaixo), 25 mL de d-Biotina estoque (4000 x estoque, 40 M em PBS armazenados como alíquotas a -20 ° C. Sigma B4639), 100 ml de insulina (1000 x estoque, 5 mg / ml em HCl 0.01N armazenados como alíquotas a -20 ° C, Sigma 16.634), e 1 ml de penicilina / estreptomicina (100 x estoque, Invitrogen 15140), filtro de esterilizar e armazenar a 4 ° C. Sato 100 x solução estoque: Para fazer 40 ml do estoque: misturar 40 ml Neurobasal com 400 mg apo-transferrina (Sigma T2252), 400 mg BSA (Sigma A9647), 10 ml de progesterona (25 mg de etanol / ml, armazenado como alíquotas a -20 ° C. Sigma P8783), 64 mg putrescina (Sigma P7505), e 40 mL de selenito de sódio (30 mM em PBS, armazenado como alíquotas a -20 ° C, Sigma S5261). Filtro de esterilizar a solução estoque Sato, e armazenar como alíquotas a -20 ° C. Neurônio médio chapeamento (PM): Médio Neurobasal, 2% B27, 2mM de glutamina (stock 100x, alíquotas armazenadas a -20 ° C, Invitrogen 25030), 25μM de ácido glutâmico (100 ações, alíquotas armazenadas a -20 ° C) e 1% penicilina estreptomicina /. Neurônio meio de cultura (CM): Médio Neurobasal, 2% B27, 2mM de glutamina (stock 100x, alíquotas armazenadas a -20 ° C) e 1% de penicilina / estreptomicina. 5 FDU estoque: estoque 100x, 1mM 5-fluoro-2&#39;-deoxiuridina (Sigma F0503) and1mM uridina (Sigma U3003) em meio Neurobasal. Esterilizado por filtração e armazenado como alíquotas a -20 ° C. Papaína solução digestão (fazer logo antes do dissecção): <ol><li> Dissolver 3,2 mg L-Cisteína (Sigma C-7352) em 4ml DM </li><li> Ajustar o pH a cerca de 7,4 com NaOH 1N (teste em tiras de teste pH) e coloque em banho-maria a 37 ° C Nota: Execute o próximo passo direito, antes da digestão dos tecidos (veja abaixo). </li><li> Adicionar papaína para uma concentração final de 20 unidades / ml </li><li> Filtro de esterilizar (0,22 milímetros), e colocar em banho-maria a 37 ° C </li></ol> Tripsina solução inibidora (fazer logo antes do dissecção): <ol><li> Dissolver 0,2 g inibidor de tripsina (Sigma T7295) no DM 20ml </li><li> Verificar o pH e ajustar o pH a 7,4 com 1 ~ N NaOH </li><li> Filtro de esterilizar e colocar em banho-maria a 37 ° C </li></ol> Dissecação cérebro Configuração: <ul><li> Pinça esterilizada e tesoura </li><li> Etanol 70% </li><li> Lenço de papel limpo e DIAPer almofada no banco </li><li> Placas de Petri com 10 centímetros gelada DM para o útero eo feto no banco </li><li> Plataforma de gelo em microscópio de dissecção </li><li> 2-3 pratos seis centímetros com DM no gelo </li><li> PM quente no banho-maria a 37 ° C </li></ul><ol><li> Euthanize uma grávida de ratos Sprague-Dawley (E16) de acordo com o procedimento aprovado pelo Institutional Animal Care and Us Committee (IACUC) </li><li> Coloque o rato sobre o bloco de fralda e álcool spay na região abdominal </li><li> Use uma pinça para segurar a pele abdominal e um par de tesouras para fazer uma incisão forma de V cortando apenas a pele </li><li> Afastados da pele e usar um outro pinças esterilizadas e tesouras para cortar a camada muscular </li><li> Extrato da cadeia dos sacos embrião e transferi-los para um prato contendo 10 centímetros gelada DM </li><li> Usando uma tesoura de microdissecção, remova cuidadosamente cada embrião a partir do saco eo cérebro de cada embrião, e colocar o cérebro livre em um prato limpo, com 10 centímetros DM em uma plataforma de gelo </li><li> Em uma capa Laminar e sob um microscópio de dissecção, remova mesencéfalo / rombencéfalo seções, e utilize uma pinça fina para remover meninges, e transferir os córtices limpos / hipocampo para um novo prato com seis centímetros DM em um bloco de gelo Nota: nesta fase, o tecido está pronto para a digestão enzimática. </li><li> Faça papaína e tripsina solução inibidora e filtro de esterilizar </li><li> Remover o excesso de córtices DM / hipocampos </li><li> Adicionar 4ml da solução preparada papaína digestão </li><li> Transferência de todo o conteúdo para um tubo de 50ml e colocar o tubo Falcon em banho-maria a 37 ° C por exatamente 5 minutos </li><li> Remoção da solução de papaína com pipeta </li><li> Adicionar solução inibidora de tripsina 5ml, redemoinho do tubo, e coloque em banho-maria a 37 ° C por 2-3 min </li><li> Remover solução inibidora de tripsina </li><li> Repita o passo 13 e 14 três vezes </li><li> Adicionar PM morna 20ml </li><li> Triturar até que todos os grumos desapareceram (~ 20-30 vezes) </li><li> Centrifugar a 100xg para 7min </li><li> Ressuspender o sedimento em 10ml PM e lavar duas vezes por centrifugação </li><li> Ressuspender o sedimento em 10ml PM para as culturas de neurônios regular ou em NBB27/DMEM para culturas agregadas </li><li> Passe as células através de uma peneira 70μm celular </li><li> Contagem de células vivas </li><li> Proceder às culturas agregadas, como descrito abaixo ou a placa de células para as culturas de neurônios regulares Nota: Nesta etapa, as culturas de neurônios altamente enriquecido pode ser alcançado por plaqueamento das células dissociadas em PDL revestimento de placas a densidade de 200-640 células / mm 2, dependendo da finalidade de experimentos. Depois que as células tenham aderido (~ 2-4h), substituir média com PM quente. Se cultivo por longos períodos de tempo, no dia 4, substitua a metade da média com CM quente. Para culturas altamente purificada neuronal, inibidor mitótico 5 FDU (concentração final 10 mM) podem ser adicionados a div2 para inibir a não-proliferação de células neuronais (ie, durante div2-3). Após a recuperação no CM por 2 dias, as células são tratadas de pulso novamente com 5-FDU por mais 2 dias (DIV 6-7) seguido por uma mudança médio completo. Depois, substituir metade do meio com frescos, CM morna a cada 3-4 dias. Neurônios são viáveis ​​por até quatro semanas. </li></ol> Agregados preparação e revestimento <ol><li> Suspender células dissociadas em meio NBB27/DMEM contendo 1x Sato, 10ng/ml CNTF e 10μM forskolin a uma densidade de 2x10 6 células / ml </li><li> Transferência de 2 ml de suspensão de células em cada poço de uma placa não revestidos 6-bem </li><li> Cultura para 3 noites. Cada dia, gentilmente ressuspender as células, uma vez usando um Pipetman P1000 Nota: As células começam a formar agregados (Figura 1). </li><li> No dia antes do plaqueamento agregado, casaco placa / lamínulas com Matrigel: <ul><li> Diluir estoque Matrigel (fator de crescimento reduzido, BD Biosciences 354230) 1:20 com DMEM frio no gelo Nota: alíquotas Matrigel deve ser preparado por protocolo fabricante. Todas as dicas, os tubos de Eppendorf e soluções para a tomada de alíquotas do estoque deve ser frio para evitar a polimerização Matrigel. Alíquotas de Matrigel são mantidos a -80 ° C e descongeladas a 4 ° C. </li><li> Brasão anteriormente PDL revestido com lamínulas 300μl/well da solução diluída Matrigel durante a noite em uma incubadora de 37 ° C </li><li> Lavar com PBS quente, em seguida, lave com água morna estéril placa DDH 2 O e deixar as placas na incubadora. </li></ul></li><li> 3 dias após a formação de agregados, delicadamente ressuspender as células novamente e peneirar a suspensão através de uma malha 200μm em um tubo Falcon de 50ml. Permitir agregados para resolver para o fundo do tubo por gravidade (~ 3-5min) </li><li> Remova cuidadosamente o sobrenadante </li><li> Adicionar médio, delicadamente ressuspender os agregados e deixá-los resolver novamente. Repita este procedimento várias vezes para remover as células mortas, as células individuais e detritos. Uso de microscópio para verificar se o sobrenadante ainda conter não agregados células e debris </li><li> Delicadamente ressuspender os agregados no meio NBB27/DMEM mesmos (Figura 1) e contagem de agregados </li><li> Ajustar a densidade de agregados para cerca de 25-30 aggreggats/50ul </li><li> Alíquotas de transferência (500-1000μl) da suspensão total para tubos Eppendorf de 2 ml para revestimento Nota: Como agregados tendem a estabelecer-se no fundo dos tubos, é crucial para fazer tubos múltiplos da suspensão agregado para revestimento. Delicadamente ressuspender os agregados muitas vezes antes da semeadura-los em lamínulas revestidos ou placas de cultura para garantir número similar de agregados banhado em cada lamela. </li><li> Retire a placa de cultura contendo Matrigel revestido lamínulas de incubadora. Remoção da solução de cada bem, lavar as lamelas com PBS quente e 2 DDH O, e, em seguida, uma breve e seco. Eles estão agora prontos para a semeadura agregado </li><li> Inverter um tubo contendo a suspensão total, que lhes permitam ser distribuídos uniformemente antes da semeadura. 50μl de suspensão de carga agregada no centro de cada lamela, e gentilmente colocou a placa de volta à incubadora, sem qualquer perturbação adicional para permitir agregados para anexar uniformemente para a lamela Nota: A densidade dos agregados é crucial. Se agregados são semeadas muito próximos uns dos outros ou se a densidade de semeadura é alta, eles tendem a se fundem à medida que crescem. </li><li> Adicionar DMEM/NBB27medium adicionais (500 mL) a cada 4-6 h bem mais tarde ou cedo na manhã seguinte </li><li> Para manter a cultura agregado, metade mudança do meio a cada 3-4 dias. Quando a mudança médio, ter cuidado para não perturbar os agregados e isso é feito por meio suavemente adicionando ao longo da parede lateral do poço. Nota: Poucas horas depois de agregados ligados à placa, crescimento de neuritos dos agregados pode ser observada. Axônios crescem rapidamente nas primeiras duas semanas e formar conexões abundantes entre os agregados (Figura 2). Progenitores gliais migram radialmente para fora dos agregados e diferenciar ao longo do tempo em astrócitos e oligodendrócitos maduros. </li></ol><img src="/files/ftp_upload/1304/1304fig1.jpg" alt="figura 1" /> Figura 1. Imagens de contraste de fase de células formando agregados em dias diferentes e agregados após a lavagem. <img src="/files/ftp_upload/1304/1304fig2.jpg" alt="figura 2" /> Figura 2. Phase imagens de contraste de uma cultura agregada 2 e 12 dias após semeado em Matrigel revestido lamínulas.

<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>

Os primeiros estudos relataram a formação de sinapses e de mielina maduras em rotação mediada livre de culturas agregada flutuante 1. O SNC sistema de cultura agregada descrita aqui combina sem soro crescimento de células progenitoras multipotentes em três agregados dimensional com a conveniência de culturas tradicionais em 2D para facilitar a análise de migração celular, desenvolvimento e diferenciação in vitro. O sistema pode ser modificado e usado para precursoras neurais pesquisas com...

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

Um sistema in vitro que recapitula o desenvolvimento e diferenciação de progenitores em neurônios maduros e células gliais no sistema nervoso central (SNC) proporcionaria uma plataforma poderosa para os neurocientistas para investigar axo-glial interações, propriedades e diferenciação de células progenitoras multipotentes, ea progressão da oligodendroglial células da linhagem no nível celular e molecular. Descrevemos aqui um sistema de cultura CNS agregado de prosencéfalo embrionárias de ratos, que podem ser mantidos em um meio isento de soro até 3-4 semanas e é usado em nosso laboratório como modelo para estudar a interação neurônio glia e mielinização do SNC. Este vídeo vai demonstrar como isolar e cultivar essas culturas CNS agregado de E16 cérebro de rato. Além disso, a partir da dissecção mesmo cérebro, altamente enriquecido regulares dissociada culturas neuronais podem ser facilmente obtidos e utilizados para vários estudos sobre os neurônios do SNC ou utilizados para co-culturas com outras células.

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 protocolos para um sistema cerebral de ratos embrionários cultura agregada é descrita. Progenitores multipotentes nos agregados podem se desenvolver e se diferenciar em neurônios, astrócitos e oligodendrócitos.

Preparação das Culturas cérebro de um rato Aggregate para Neuron Glia e Estudos de Desenvolvimento

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 visualizações.  Texas A&M University (TAMU). A protocolos para um sistema cerebral de ratos embrionários cultura agregada é descrita. Progenitores multipotentes nos agregados podem se desenvolver e se diferenciar em neurônios, astrócitos e oligodendrócitos.

Vídeo: 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.

Culturas primárias neuronais do cérebro de Tarde Stage Drosophila pupas

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

Biologia

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.

Preparação de culturas de neurônios a partir de embriões Midgastrula Stage Drosophila

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: Mecanismo e desenvolvimento de novas terapias

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.

Fabricação de um dispositivo micro para a compartimentação de Neuron Soma e axônios

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.

Preparação de Dissociated rato Culturas Neuron Cortical

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.

Preparação de 2-dGuo-Tratada Culturas Organ 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.

Preparação de Aplysia Sensório-motor Neuronal culturas de células

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.

Um compartimento Multi-glia do SNC Neuron-Co-cultura plataforma microfluídica

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.

Indução toxina e Extração de Proteínas de Fusarium Spp. Culturas de Estudos Proteômica

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.

Preparação de tendões da cauda de rato de Estudos biomecânicos e Mechanobiological

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