Este trabalho foi financiado pelo NIH para conceder NS27501 DKOD. Suporte adicional para este trabalho foi fornecida por uma concessão para a UC Irvine em apoio DKOD através do Programa Professor HHMI.

Preparações antes do dia da cultura: <ol><li> Fazer a solução de dissecar estéril. </li><li> Faça DMEM estéril e manter em alíquotas de 10 ml a 4 ° C por 2 semanas. </li><li> Tornar estéril DDM2 suplementos e congelar em 50 ou 100 mL alíquotas de 1 mês. </li><li> Faça ConA / laminina. </li><li> Lamínulas casaco. Opcional: Faça CNBM estéril e armazenar congelado por até 4 meses. </li></ol> No dia da cultura I. Preparar solução enzimática (ES), em capela de fluxo laminar <ol><li> Coloque 5 ml de solução de dissecação (DS) em um tubo de centrífuga de 15 ml. </li><li> Pesar 0,8 mg de L-cisteína em um eppendorf 0,6 ml e adicionar 150 mL de DS. Pipeta para cima e para baixo até que os cristais são ido. </li><li> Adicione 150 ml de DS / Cisteína aos 5 DS ml e misture bem. </li><li> Adicionar 50 U (unidades) de papaína. </li><li> Adicionar 7 ml de 0,1 N Na OH e misture bem. Solução clara dentro de 30 min. quando ativado. </li><li> Solução enzimática de filtro com 0,2 milímetros filtro de seringa de 1,5 ml em um tubo de microcentrífuga estéril parafuso cap Papaína: Quer 50 U em 5 ml DS. Cada lote é um pouco diferente, por isso deve calcular quanto: 37,3 mg / ml e 28,3 U / mg 37,3 x 28,3 = 1055,59 U / ml (1000 ml) 50 U = 47,4 ml </li></ol> II. Faça DDM2 media No dia da cultura: adicione os seguintes suplementos para fazer DDM2 A 10 ml de DMEM adicionar os suplementos, pouco antes de cultivo: <ol><li> 100μl Transferrina </li><li> 100μl Putrescine </li><li> 100μl Selenium </li><li> Progesterona 100μl </li><li> Insulina 50μl </li><li> 10μl 20-Hydroxyecdysone </li></ol> Nota: Faça o suficiente para todas as culturas DDM2 planejado (cada cérebro banhado em uma lamela em um único prato de 35 milímetros individuais Petri, 1,5 ms dos meios de comunicação / cultura) Etapa III-VI pode estar em uma bancada de laboratório feito não estéreis e devem ser concluídas em 45 minutos ou menos. III. Pupas Colecção <ol><li> Selecione 15/10 pupas de lados do frasco com um microscópio de dissecação. </li><li> Pupas lugar na tampa de um prato de 35 milímetros seco Petri. </li></ol> Nota: Nós usamos geralmente cérebros de pupas entre 55-78 horas após a formação pupário (APF). É ligeiramente mais difícil de dissecar o cérebro dos mais jovens pupas, as cápsulas cabeça tendem a entrar em colapso, enquanto o nível geral de crescimento de neuritos é ligeiramente mais baixa desde o mais velho pupas. Na cepa do tipo selvagem Canton-S, estes estágios são reconhecidos pelos olhos que são pigmentadas castanho claro a ligeiramente avermelhado, com pouco pigmento em outros lugares. IV. Decapitação sob microscópio de dissecação <ol><li> Coloque duas gotas de solução estéril em dissecar tampa da placa de Petri, ao lado de pupas. </li><li> Suavemente realizar uma única pupa com uma pinça fina na mão esquerda e usar um medidor de 27 agulha da seringa conectada a uma seringa cc 1 na mão direita para remover a cutícula na extremidade anterior de pupa. </li><li> Squeeze pupa ligeiramente com uma pinça e emerge como chefe use agulha de calibre 27 para cortar o pescoço. </li><li> Com a ponta da agulha ou a pinça, a transferência de cabeça para queda de dissecar solução. </li><li> Repita até que você tenha 10-15 cabeças em uma única gota. </li></ol> V. A remoção do cérebro de cabeça sob microscópio de dissecação <ol><li> Em cada mão segurar uma seringa de 1 cc com uma agulha de calibre 27. </li><li> Use-os para a posição da cabeça voar em uma gota de solução para dissecar a probóscide é voltado para você ea superfície dorsal da cabeça é o norte. </li><li> Inserir a agulha esquerda para a probóscide para fixar a cabeça para baixo e use a agulha direita para fazer uma fenda no olho direito que vai da superfície ventral para dorsal. </li><li> Inserir a agulha direita perto do lado esquerdo na região da tromba e depois usar a agulha esquerda para a acione a cutícula sobre o olho esquerdo, empurrando o cérebro na direção da fenda no olho direito. O cérebro deve emergir da fenda do lado direito, relativamente intacta, muitas vezes com ambos eis ópticabes ainda ligado e por vezes, o tecido ocular, bem pigmentada. </li><li> Empurrar o cérebro na parte traseira da agulha e transfira para uma gota de solução salina estéril em dissecar um novo estéril 35 mm, placa de Petri. </li><li> Coletar 15/10 cérebros para cada genótipo. </li></ol> VI. Remoção dos lobos óptica e tratamento enzimático <ol><li> Em cada mão segurar uma seringa de 1 cc com uma agulha de calibre 27 e usá-los para coletar todos os cérebros em uma pequena área da gota de solução de dissecação </li><li> Use seringas como ferramentas de corte para remover os lobos ótica de cada um cérebro de modo que o tecido remanescente para a cultura é a região do cérebro central. </li><li> Use um Pipetman 20 l, fixado em 5 mL, para transferir todos os cérebros de um genótipo único para um tubo eppendorf contendo 1 ml de solução de papaína estéril. </li><li> Cérebros incubadas em papaína por 10-15 minutos em um conjunto rotador em 60 rpm. </li><li> Centrifugar a 3000 rpm por 3 minutos. </li></ol> Dentro capô estéril de fluxo laminar - todas as etapas onde o tecido é exposto ao ar deve ser feito em capela de fluxo laminar para as etapas restantes. VII. Lavagem <ol><li> Remover e substituir solução enzimática com solução estéril de dissecação. </li><li> Centrifugar a 3000 rpm por 3 minutos. </li><li> Lave com uma solução estéril dissecar um total de 3 vezes. </li><li> Remover salina dissecar e substituir com DDM2. </li><li> Centrifugar e lavar um total de 2 vezes com DDM2. </li><li> Transferência de cérebros e de mídia para um prato de 35 milímetros estéril Petri. </li></ol> VIII. Trituração e Plating sob microscópio de dissecação <ol><li> Lugar de 5 ul DDM2 no centro de uma lamela. </li><li> Transferência de um cérebro para essa queda de DDM2 com uma ponta amarela. </li><li> Sob o microscópio, use 27 agulhas de dados até o cérebro em pedaços pequenos (devem ter &lt;1 minuto). </li><li> Sob a orientação visual, quebrar a ponta de uma pipeta de vidro que foi puxado para uma ponta fina para que as peças maiores podem ser delicadamente sugado para a ponta da pipeta e expulsos de volta para o meio. Usamos tubos de boca de sucção que vêm de fábrica com 100 mL de vidro hematócrito capilar. Nota: Tome cuidado para não ficar bolhas na mídia e você precisará determinar empiricamente a pipeta melhor tamanho para usar. Bons permitem dissociar o cérebro com algumas células individuais e ainda algumas grandes aglomerações, em cerca de 30 segundos / cérebro. Quando você olha para estes em maior potência, aproximadamente 10-20% dos neurônios ainda deve ter alguns processos remanescentes e ainda haverá alguns grandes aglomerações. Se você dissociar demais, então todas as células serão rodada e terão sofrido danos tanto que eles não vão sobreviver. Esta etapa requer prática e deve ser feito rapidamente, pois há uma relação superfície-volume grande ea osmolalidade e pH da gota de DDM2 podem mudar rapidamente. Tecido precisa ser colocado em incubadora de CO 2 o mais rápido possível. </li><li> Escrever sobre a tampa placa de Petri: &quot;O genótipo, data e hora&quot;. </li><li> Coloque a placa de Petri 35 milímetros em um prato de 100 milímetros de Petri (com kimwipe molhado) e deixe se instalar na incubadora de CO 2 por 30 min. </li><li> Inundar o prato de 35 mm com 1,5 ml de DDM2. </li><li> Manter culturas na incubadora a 5% incubadora de CO 2 (23 ° C). Nota: Se você não tem acesso a um resfriamento incubadora de CO 2, use um padrão de cultura de tecidos de mamíferos incubadora e quer colocá-lo em uma sala fria e calor a 23 ° C, ou colocar no laboratório, desligue temp, e usar gelo em uma bandeja na parte inferior da incubadora para regular a temperatura em torno de ambiente. </li></ol> IX. Células de alimentação <ol><li> No 1 º dia (dia seguinte), adicionar 0,5 ml de CNBM/B27 (Media condicionado) para fazer 03:01 DDM2: CNBM/B27. </li><li> No dia 5 substituir 1 ml de mídia para 03:01 DDM2: CNBM/B27. </li><li> Manter as células de alimentação com 03:01 DDM2: CNBM/B27 cada 4-5 dias. Nota: As culturas podem ser cultivadas sem a mídia não-neuronal condicionado, mas os neurônios parecem um pouco mais frágil e são mais difíceis para uso em experimentos de eletrofisiologia. </li></ol>

<ol>
	<li>Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culturing Nerve Cells. , (1991).</li><li>Rohrbough, J., O'Dowd, D. K., Baines, R. A., Broadie, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+bases+of+behavioral+plasticity:+Establishing+and+Modifying+synaptic+circuits+in+the+Drosophila+Genetic+System.">Cellular bases of behavioral plasticity: Establishing and Modifying synaptic circuits in the Drosophila Genetic System.</a> J. Neurobiol. 54, 254-271 (2003).</li><li>O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage-gated+currents+and+firing+properties+of+embryonic+Drosophila+neurons+grown+in+a+chemically+defined+medium.">Voltage-gated currents and firing properties of embryonic Drosophila neurons grown in a chemically defined medium.</a> J. Neurobiol. 27, 113-126 (1995).</li><li>Lee, D., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+excitatory+synaptic+transmission+mediated+by+nAChR+in+cultured+Drosophila+neurons.">Fast excitatory synaptic transmission mediated by nAChR in cultured Drosophila neurons.</a> J. Neurosci. 19, 5311-5321 (1999).</li><li>Su, H., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+synaptic+currents+in+Drosophila+mushroom+body+Kenyon+cells+are+mediated+by+alpha-bungarotoxin-sensitive+nAChRs+and+picrotoxin-sensitive+GABA+receptors.">Fast synaptic currents in Drosophila mushroom body Kenyon cells are mediated by alpha-bungarotoxin-sensitive nAChRs and picrotoxin-sensitive GABA receptors.</a> J. Neurosci. 23, 9246-9253 (2003).</li><li>Jiang, S. A., Campusano, J. M., Su, H., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+mushroom+body+Kenyon+cells+generate+spontaneous+calcium+transients+mediated+by+PLTX-sensitive+calcium+channels.">Drosophila mushroom body Kenyon cells generate spontaneous calcium transients mediated by PLTX-sensitive calcium channels.</a> J. Neurophysiol. 94, 491-500 (2005).</li><li>Campusano, J. M., Su, H., Jiang, S. A., Sicaeros, B., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=nAChR-mediated+calcium+responses+and+plasticity+in+Drosophila+Kenyon+cells.">nAChR-mediated calcium responses and plasticity in Drosophila Kenyon cells.</a> Develop Neurobiol. 67, 1520-1532 (2007).</li><li>Oh, H. -. W., Campusano, J. M., Hilgenberg, L. G. W., Sun, X., Smith, M. A., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+analysis+of+chemical+synapses+and+gap+junctions+between+Drosophila+brain+neurons+in+culture.">Ultrastructural analysis of chemical synapses and gap junctions between Drosophila brain neurons in culture.</a> Develop Neurobiol. , (2007).</li></ol>

Neurônios colhidos os cérebros de roedores embrionárias / pós-natal podem ser cultivadas em cultura de células primária onde se estendem neurites e formam conexões sinápticas funcionais. Métodos para a preparação dessas culturas estão bem estabelecidas e estudos em culturas de roedores neuronal têm desempenhado um papel fundamental na identificação de genes e fatores ambientais envolvidos na regulação da formação de sinapses e função (Banker e Goslin, 1991). Enquanto os neurônios do inseto a partir de uma variedade de ...

Lamelas revestimento: Coloque lamínulas autoclavado numa placa de Petri 60 mm. Ul pipeta 5 de Con A / mix laminina em centro de cada lamela. Lugar em 37 C incubadora por 2 horas. Enxágüe lamínulas 3x com 100 ul de água autoclavada cada. Use a vácuo ligado a uma pipeta Pasteur estéril. Durante o último enxaguamento, pegar a lamela com uma pinça e seco ambos os lados. Transferência a lamela e uma placa de Petri de 35mm. Conservar à temperatura ambiente por até um mês.

primary neuronal cultures from the brains of late stage drosophila pupae

Neste vídeo, demonstramos a preparação de culturas primárias de neurônios do cérebro de final de estágio Drosophila pupas. O procedimento começa com a remoção dos cérebros de animais em 70-78 horas após a formação pupário. Os cérebros isolados são mostrados após incubação em breve papaína seguido por várias lavagens em soro livre de meio de crescimento. O processo de dissociação mecânica de cada cérebro em uma queda de 5 ul de mídia em uma lamela é ilustrado. Os axônios e dendritos dos neurônios pós-mitóticas são cisalhado off perto da soma durante a dissociação, mas os neurônios começam a regenerar os processos dentro de algumas horas de placas. Imagens mostram culturas vivas em dois dias. Neurônios continuar a desenvolver processos durante a primeira semana de cultura. Populações neuronais específicas podem ser identificadas em cultura usando GAL4 linhas a dirigir expressão tecido específico de marcadores fluorescentes, como GFP ou RFP. Gravações de células inteiras têm demonstrado que os neurônios cultivados forma funcional, espontaneamente ativa sinapses colinérgicas e GABAérgica. Um segmento pequeno vídeo ilustra a dinâmica do cálcio nos neurônios cultivados usando Fura-2 como um corante indicador de cálcio para monitorar transientes de cálcio espontânea e nicotina respostas evocadas de cálcio em um prato de neurônios cultivados. Estas culturas cérebro pupal são um sistema modelo útil em que as ferramentas genéticas e farmacológicas podem ser usados ​​para identificar os fatores intrínsecos e extrínsecos que influenciam a formação ea função de sinapses central.

Watch this Scientific Journal Video about Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae at JoVE.com

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

Este vídeo demonstra a preparação de culturas primárias de neurônios do cérebro de final de estágio Drosophila pupas. Pontos de vista de culturas vivas mostram crescimento de neuritos e imagem dos níveis de cálcio usando 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 ...

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Biology

15.0K visualizações.  University of California, Irvine (UCI). Este vídeo demonstra a preparação de culturas primárias de neurônios do cérebro de final de estágio Drosophila pupas. Pontos de vista de culturas vivas mostram crescimento de neuritos e imagem dos níveis de cálcio usando Fura-2.

Vídeo: Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

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 ...

Biologia

Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information into directional movement towards the appropriate target cell. To fully understand how guidance information is transmitted from the cell surface to the underlying dynamic cytoskeletal networks, one needs a model system suitable for live cell imaging of protein dynamics at high temporal and spatial resolution. Typical vertebrate growth cones are too small to quantitatively analyze F-actin and microtubule dynamics. Neurons from the sea hare Aplysia californica are 5-10 times larger than vertebrate neurons, can easily be kept at room temperature and are very robust cells for micromanipulation and biophysical measurements. Their growth cones have very defined cytoplasmic regions and a well-described cytoskeletal system. The neuronal cell bodies can be microinjected with a variety of probes for studying growth cone motility and guidance. In the present protocol we demonstrate a procedure for dissection of the abdominal ganglion, culture of bag cell neurons and setting up an imaging chamber for live cell imaging of growth cones.

Aplysia californica neurons develop large growth cones in culture that are excellent for high-resolution imaging of growth cone motility and guidance. Here, we present a protocol for dissection and plating of Aplysia bag cell neurons as well as for setting up a chamber for live cell imaging.

Culturas de células neuronais de Aplysia para imagens de alta resolução de Cones de Crescimento

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information ...

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

Principal Dissociated Midbrain dopamina culturas de células de recém-nascidos de roedores

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

Elaboração de culturas de células primárias hematopoéticas de medula óssea de camundongos para Eletroporação

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

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 ...

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three preparations of the brain and visual system that cover the range from the developing eye disc-brain complex in the developing pupae to individual eye and brain dissection from adult flies. All protocols are optimized for the live culture of the preparations. However, we also present the conditions for fixed tissue immunohistochemistry where applicable. Finally, we show live imaging conditions for these preparations using conventional and resonant 4D confocal live imaging in a perfusion chamber. Together, these protocols provide a basis for live imaging on different time scales ranging from functional intracellular assays on the scale of minutes to developmental or degenerative processes on the scale of many hours.

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.

Preparação de Desenvolvimento e Adultos Drosophila Brains e retina de imagens ao vivo

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

Establishing Primary Adult Fibroblast Cultures From Rodents

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel.

This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.

Estabelecer culturas de fibroblastos adultos primária de roedores

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

Culturas de células primárias de Drosophila Os embriões Gástrula

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

Naïve Adult Stem Cells isolamento de culturas primárias de fibroblastos humanos

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This methods paper describes how to isolate mid-to-late stage follicles (Stage 10B-14) and utilize them to provide new insights into the molecular and morphologic events occurring during tight windows of developmental time. Isolated follicles can be used for a variety of experimental techniques, including in vitro development assays, live imaging, mRNA expression analysis and western blot analysis of proteins. Follicles at Stage 10B (S10B) or later will complete development in culture; this allows one to combine genetic or pharmacologic perturbations with in vitro development to define the effects of such manipulations on the processes occurring during specific periods of development. Additionally, because these follicles develop in culture, they are ideally suited for live imaging studies, which often reveal new mechanisms that mediate morphological events. Isolated follicles can also be used for molecular analyses. For example, changes in gene expression that result from genetic perturbations can be defined for specific developmental windows. Additionally, protein level, stability, and/or posttranslational modification state during a particular stage of follicle development can be examined through western blot analyses. Thus, stage-specific isolation of Drosophila follicles provides a rich source of information into widely conserved processes of development and morphogenesis.

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Stage-specific isolation of mid-to-late Drosophila follicles is useful for a variety of purposes. Such follicles develop in culture, which allows for genetic and/or pharmacologic manipulations to be coupled with in vitro development assays and live imaging. Additionally, follicles can be used for molecular studies, such as isolating mRNA and protein.

O Utilitário de Estágio específico-Mid-de-final<em&gt; Drosophila</em&gt; Folículo Isolamento

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This ...