Gostaríamos de agradecer aos membros do grupo Potter para fornecer observações inestimáveis ​​sobre o protocolo. Este trabalho foi financiado por uma bolsa de investigação clínica de treinamento da Academia Americana de Neurologia da Fundação para CMH, uma bolsa de estudos do Instituto Nacional de Ciências Médicas Gerais (NIGMS; <a href="http://www.nigms.nih.gov/">http://www.nigms.nih.gov/</a> ) para JDR (GM08169 ), uma concessão do National Institute of NIH Distúrbios Neurológicos e Derrame (NS054809), uma Ruth L. Kirschstein Prêmio Nacional de Serviço de Pesquisa de JDR (NS060392), um NSF Efri subvenção (0.836.017), a Fundação Coulter Prêmio de Pesquisa Translacional, e um bolsa de pesquisa translacional para JDR (NS007480).

<ol>
	<li>Potter, S. M., DeMarse, T. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+approach+to+neural+cell+culture+for+long-term+studies.">A new approach to neural cell culture for long-term studies.</a> J Neurosci Methods. 110, 1-2 (2001).</li><li>Rambani, K., Vukasinovic, J., Glezer, A., Potter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+thick+brain+slices:+an+interstitial+3D+microperfusion+system+for+enhanced+viability.">Culturing thick brain slices: an interstitial 3D microperfusion system for enhanced viability.</a> J Neurosci Methods. 180, 243-254 (2009).</li><li>Bakkum, D. J., Chao, Z. C., Potter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatio-temporal+electrical+stimuli+shape+behavior+of+an+embodied+cortical+network+in+a+goal-directed+learning+task.">Spatio-temporal electrical stimuli shape behavior of an embodied cortical network in a goal-directed learning task.</a> J Neural Eng. 5, 310-323 (2008).</li><li>Shahaf, G., Marom, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learning+in+networks+of+cortical+neurons.">Learning in networks of cortical neurons.</a> J Neurosci. 21, 8782-8788 (2001).</li><li>Wagenaar, D. A., Nadasdy, Z., Potter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistent+dynamic+attractors+in+activity+patterns+of+cultured+neuronal+networks.">Persistent dynamic attractors in activity patterns of cultured neuronal networks.</a> Phys Rev E Stat Nonlin Soft Matter Phys. 73, 051907-05 (2006).</li><li>Esposti, F., Signorini, M. G., Potter, S. M., Cerutti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statistical+long-term+correlations+in+dissociated+cortical+neuron+recordings.">Statistical long-term correlations in dissociated cortical neuron recordings.</a> IEEE Trans Neural Syst Rehabil Eng. 17, 364-369 (2009).</li><li>Madhavan, R., Chao, Z. C., Potter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+of+recurring+spatiotemporal+activity+patterns+in+cortical+networks.">Plasticity of recurring spatiotemporal activity patterns in cortical networks.</a> Phys Biol. 4, 181-193 (2007).</li><li>Chiappalone, M., Massobrio, P., Martinoia, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Network+plasticity+in+cortical+assemblies.">Network plasticity in cortical assemblies.</a> Eur J Neurosci. 28, 221-237 (2008).</li><li>Hofmann, F., Bading, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+term+recordings+with+microelectrode+arrays:+studies+of+transcription-dependent+neuronal+plasticity+and+axonal+regeneration.">Long term recordings with microelectrode arrays: studies of transcription-dependent neuronal plasticity and axonal regeneration.</a> J Physiol Paris. 99, 2-3 (2006).</li><li>Gortz, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+reduction+of+spontaneous+neuronal+network+activity+by+sublethal+amyloid+beta+(1-42)+peptide+concentrations.">Transient reduction of spontaneous neuronal network activity by sublethal amyloid beta (1-42) peptide concentrations.</a> J Neural Transm. 116, 351-355 (2009).</li><li>Koito, H., Li, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+rat+brain+aggregate+cultures+for+neuron+and+glia+development+studies.">Preparation of rat brain aggregate cultures for neuron and glia development studies.</a> J Vis Exp. 31, (2009).</li><li>Brewer, G. J., Cotman, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+and+growth+of+hippocampal+neurons+in+defined+medium+at+low+density:+advantages+of+a+sandwich+culture+technique+or+low+oxygen.">Survival and growth of hippocampal neurons in defined medium at low density: advantages of a sandwich culture technique or low oxygen.</a> Brain Res. 494, 65-74 (1989).</li><li>Lelong, I. H., Petegnief, V., Rebel, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+cells+mature+faster+on+polyethyleneimine+coated+plates+than+on+polylysine+coated+plates.">Neuronal cells mature faster on polyethyleneimine coated plates than on polylysine coated plates.</a> J Neurosci Res. 32, 562-568 (1992).</li><li>Potter, S. M., Pine, J., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+transplant+staining+with+DiI+and+vital+imaging+by+2-photon+laser-scanning+microscopy.">Neural transplant staining with DiI and vital imaging by 2-photon laser-scanning microscopy.</a> Scanning Microsc Suppl. 10, 189-199 (1996).</li><li>Rolston, J. D., Gross, R. E., Potter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+low-cost+multielectrode+system+for+data+acquisition+enabling+real-time+closed-loop+processing+with+rapid+recovery+from+stimulation+artifacts.">A low-cost multielectrode system for data acquisition enabling real-time closed-loop processing with rapid recovery from stimulation artifacts.</a> Front Neuroengineering. 2, (2009).</li><li>Wagenaar, D. A., Potter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+versatile+all-channel+stimulator+for+electrode+arrays,+with+real-time+control.">A versatile all-channel stimulator for electrode arrays, with real-time control.</a> J Neural Eng. 1, 39-45 (2004).</li><li>van Pelt, J., Wolters, P. S., Corner, M. A., Rutten, W. L., Ramakers, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+characterization+of+firing+dynamics+of+spontaneous+bursts+in+cultured+neural+networks.">Long-term characterization of firing dynamics of spontaneous bursts in cultured neural networks.</a> IEEE Trans Biomed Eng. 51, 2051-2062 (2004).</li><li>Wagenaar, D. A., Pine, J., Potter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+extremely+rich+repertoire+of+bursting+patterns+during+the+development+of+cortical+cultures.">An extremely rich repertoire of bursting patterns during the development of cortical cultures.</a> BMC Neurosci. 7, (2006).</li><li>Wagenaar, D. A., Potter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+multi-channel+stimulus+artifact+suppression+by+local+curve+fitting.">Real-time multi-channel stimulus artifact suppression by local curve fitting.</a> J Neurosci Methods. 120, 113-120 (2002).</li><li>Bakkum, D. J., Chao, Z. C., Potter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+activity-dependent+plasticity+of+action+potential+propagation+delay+and+amplitude+in+cortical+networks.">Long-term activity-dependent plasticity of action potential propagation delay and amplitude in cortical networks.</a> PLoS One. 3 (5), e2088-e2088 (2008).</li><li>Wagenaar, D. A., Madhavan, R., Pine, J., Potter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+bursting+in+cortical+cultures+with+closed-loop+multi-electrode+stimulation.">Controlling bursting in cortical cultures with closed-loop multi-electrode stimulation.</a> J Neurosci. 25 (3), 680-688 (2005).</li><li>Jimbo, Y., Tateno, T., Robinson, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+induction+of+pathway-specific+potentiation+and+depression+in+networks+of+cortical+neurons.">Simultaneous induction of pathway-specific potentiation and depression in networks of cortical neurons.</a> Biophys J. 76 (2), 670-678 (1999).</li><li>Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culturing nerve cells. , (1998).</li></ol>

CMH não tem nada a revelar.<br /&gt; JDR e SMP são pagos consultores para Axion Biosystems.<br /&gt; As taxas de envio e taxas de processamento de vídeo para este artigo e vídeo foram gentilmente patrocinado pela Multi-Channel Systems.

Este protocolo apresenta instruções sobre como a cultura e manter redes neuronais em micro-eletrodo de matrizes, bem como uma introdução para a gravação de redes neuronais e estimulante. Alguns dos problemas mais comuns durante a gravação de MEAs também foram discutidos na seção de solução MEA. Existem variações ocasionais no protocolo como discutido ao longo e muitas vezes essas mudanças são utilizados para otimizar as condições específicas exigidas por alguns projetos experimentais. Embora o sistema de gravação e estimular aqui apresentada é composta por nossa personalizados NeuroRighter 15 com o pré-amplificador MCS, existem vários outros sistemas que podem fornecer uma interface com as redes neuronais. Escolher uma configuração particular dependerá das necessidades específicas experimental. Gravação simplista e exemplos de estimulação foram fornecidas neste protocolo no entanto essas culturas em MEAs pode ser usado para fornecer informações sobre questões complexas sobre a atividade sináptica e conectividade de rede neuronal. Como mencionado acima, o trabalho em curso está utilizando esta tecnologia para estudar processos como plasticidade celular, excitotoxicidade, desenvolvimento e neurodegeneração. 60-10 Por exemplo, uma publicação recente de nosso laboratório mostraram reprodutíveis objetivo dirigido aprendizagem in vitro com real-tempo de estimulação de malha fechada em resposta à atividade neuronal rede atual 3. Embora a cultura MEA são muitas vezes finicky e delicado, graças à sua simplicidade e acessibilidade (em comparação com animais intactos), eles oferecem um modelo poderoso para estudar a atividade neuronal em um nível de rede.

Abastecimento <ul><li> Descartáveis ​​garrafa superior do filtro (150ml, tamanho dos poros 0,2 Hm; Nalgene # 565-0020) </li><li> Dia embrionário 18 ratos timed-fêmea grávida </li><li> Matrizes de microeletrodos (ver descrição no protocolo, B1) </li><li> Microeletrodo tampas array (ver descrição no protocolo, B3) </li><li> Nylon célula filtro (40μm, BD Falcon, # 352340) </li><li> Polipropileno cônico frascos (estéril, 15ml) </li><li> Placas de Petri de poliestireno (estéril, de 35mm e 100x15mm) </li><li> Pontas de pipeta (estéril, 20μl, 200μl e 1000μl) </li><li> Pipetas sorológicas (5ml) </li></ul> Equipamento: <ul><li> Biológicos de segurança capela de fluxo gabinete / laminar </li><li> Contador de células </li><li> Centrifugador </li><li> Recipiente de gelo </li><li> Tesouras de dissecação e pinça (esterilizada) </li><li> Dissecando microscópio em uma capela de fluxo laminar </li><li> Handheld elétrica Pipetman </li><li> Hemocitômetro </li><li> Isofluorano e câmaras de gás </li><li> Microscópio de luz </li><li> Handheld Pipetman (P-20 (20μl), P-200 (200μl), P-1000 (1000μl)) </li><li> MEA sistema de gravação (ver descrição no protocolo, D1) </li><li> Guilhotina roedores </li><li> Tri-gás incubadora (ver descrição no protocolo, B15) </li><li> Balão de vácuo, com aspirador ligado na capa </li><li> Vórtice </li><li> Banho-maria (35-37 ° C) </li></ul> Soluções e Reagentes: Solução BSA: Faça BSA 5% (Sigma A-9418) em tampão fosfato salina pH 7,4 e filtrado a 0,2 Hm. Esta solução pode ser preparado com antecedência e armazenado a 4 ° C durante vários meses. Médio célula: Combine 90 meio modificado ml Dulbecco Águia (Irvine Scientific 9024), 10 ml soro de cavalo (Gibco 16.050), piruvato de sódio 1 ml (100mM, Gibco 11.360), 250 Glutamax mL (Gibco 35.050) e insulina (concentração final 2,5 mg / ml, Sigma I-5500), em seguida, passar por um 0,2 mM Quente filter.22 e equilibrar o meio de células na incubadora antes de usar. Solução de dissecção para a preparação de solução papaína: 23 <table border="1"><tr><td> Solução de dissecção (100ml estoque) </td><td> Combine em ordem, 0,22 mM filtrar e armazenar a 4 ° C. </td><td></td></tr><tr><td> Componente </td><td> Ml de volume </td><td> Concentração Final (mM) </td></tr><tr><td> Água bidestilada </td><td> 91,175 </td><td></td></tr><tr><td> 1 M MgCl 2 </td><td> 0,58 </td><td> 5,8 mM </td></tr><tr><td> 1 M CaCl 2 </td><td> 0,025 </td><td> 0,25 mM </td></tr><tr><td> HEPES 0,5 M </td><td> 0,32 </td><td> 1,6 mM </td></tr><tr><td> 0,5% de vermelho de fenol </td><td> 0,2 </td><td> (0,001%) </td></tr><tr><td> NaOH 0,1 N </td><td> 0,2 </td><td> 0,2 mM </td></tr><tr><td> 2 M Na2SO4 </td><td> 4,5 </td><td> 90 mM </td></tr><tr><td> 1 M K2SO4 </td><td> 3,0 </td><td> 30 mM </td></tr><tr><td> 0,1 M ácido cinurênico </td><td> 1,0 </td><td> 1 mM </td></tr><tr><td> 5 mM APV </td><td> 1,0 </td><td> 0,05 mM </td></tr></table> Esta solução pode ser preparado com antecedência e armazenado a 4 ° C durante vários meses. DNAse I: Invitrogen (# 18047019). 50μl alíquotas são de flash congeladas e armazenadas a -80 ° C. Uma alíquota é descongelado no capela de fluxo laminar antes de iniciar o processo de dissociação das células. Solução salina balanceada de Hank: Sigma (# 4891). Uma solução de 1X é preparada em água deionizada estéril, filtrada em 0,2 Hm e armazenados a 4 ° C. Solução Hibernate: Prepare no dia da cultura. Combine 98 ml de meio L-15 (Invitrogen 11415), com 2 ml suplemento B-27 (50X, Invitrogen # 17504) e filtro de 0,2 mm. Solução laminina: Prepare no dia da cultura. Diluir 20μl de laminina (1mg/ml, Sigma L-2020) em 980 mL meio de células para uma concentração final de 0.02mg/ml. 20 alíquotas mL da laminina (1mg/ml) são armazenadas a -80 ° C e descongeladas imediatamente antes de usar. Descongelamento deve ocorrer na bancada e não no banho de água como o rápido degelo pode levar a gelificação da laminina. Papaína solução: 23 10 ml de solução quente dissecção (acima) para 30-32 ° C. Adicionar 200 unidades papaína (Sigma P-4762) e 1,6 mg L-cisteína (Sigma C-7880). Ajustar o pH para 7,4 com cerca de 14 mL de 0,1 N NaOH. Incubar por cerca de 30 minutos até que o cl soluçãoorelhas, filtro de 0,2 mm, flash freeze com nitrogênio líquido, em alíquotas de 2 ml em 15 ml de polipropileno estéril frascos cônicos e armazenar a -80 ° C. Descongelar uma alíquota a 35-37 ° C antes de iniciar o protocolo. Polietilenoimina solução (PEI): 13 100 ul de PEI (50% w / v, Sigma P-3143) é diluído em 100 ml tampão borato de sódio (0,1 M pH 8,4, Sigma B-9876) para uma concentração final de 0,05% w / v. Filtro estéril de 0,2 mm. Isto pode ser preparado com antecedência e armazenado a 4 ° C durante meses. Água deionizada estéril: Autoclave e deixar arrefecer à temperatura ambiente antes de usar. Tripsina (0,25% com EDTA, Gibco 25200): Alíquotas de 1ml de flash são congeladas e armazenadas a -80 ° C. Alíquotas são descongelados em banho-maria a 35-37 ° C antes do uso.

how to culture, record and stimulate neuronal networks on micro-electrode arrays (meas)

Para o século passado, muitos neurocientistas de todo o mundo têm dedicado suas vidas para entender como as redes neuronais funcionam e por que parar de trabalhar em várias doenças. Estudos incluíram observação neuropatológicos, microscopia fluorescente com rotulagem genética, e gravação intracelular em ambos os neurônios dissociados e preparações fatia. Este protocolo discute outra tecnologia, que envolve o crescimento neuronal em culturas dissociadas micro-eletrodo arrays (também chamado de multi-eletrodo arrays, AAM). Há múltiplas vantagens de usar este sistema através de outras tecnologias. Dissociada culturas neuronal em MEAs fornecer um modelo simplificado em que a atividade de rede podem ser manipulados com sequências de estimulação elétrica através de eletrodos múltiplos da matriz. Porque a rede é pequena, o impacto da estimulação é limitada a áreas observáveis, o que não é o caso de preparações intactas. As células crescem em uma monocamada fazer alterações na morfologia fácil de monitorar, com várias técnicas de imagem. Finalmente, as culturas em MEAs pode sobreviver por mais de um ano in vitro que remove todas as limitações inerentes de tempo claro com as técnicas de cultivo outras 1. Nosso laboratório e outros em todo o mundo estão utilizando esta tecnologia para fazer perguntas importantes sobre redes neuronais. O objectivo deste protocolo é fornecer as informações necessárias para a criação, cuidando, e gravação de estimular eletricamente as culturas em MEAs. Em redes vitro fornecer um meio para pedir fisiologicamente questões relevantes na rede e níveis celulares levando a uma melhor compreensão da função cerebral e disfunção.

Watch this Scientific Journal Video about How to Culture, Record and Stimulate Neuronal Networks on Micro-electrode Arrays MEAs at JoVE.com

How to Culture, Record and Stimulate Neuronal Networks on Micro-electrode Arrays MEAs

Este protocolo fornece as informações necessárias para a criação, cuidando, e gravação de estimular eletricamente as culturas na AAM.<em&gt; In vitro</em&gt; Redes de fornecer um meio para pedir fisiologicamente questões relevantes na rede e níveis celulares levando a uma melhor compreensão da função cerebral e disfunção.

Como gravar, Cultura e estimular redes neuronais em Micro-Arrays eletrodo (MEA)

For the last century, many neuroscientists around the world have dedicated their lives to understanding how neuronal networks work and why they stop ...

how-to-culture-record-stimulate-neuronal-networks-on-micro-electrode

Research

JoVE Journal

Biology

How to Culture, Record and Stimulate Neuronal Networks on Micro-electrode Arrays (MEAs)

54.6K visualizações.  Emory University School of Medicine. Este protocolo fornece as informações necessárias para a criação, cuidando, e gravação de estimular eletricamente as culturas na AAM. In vitro Redes de fornecer um meio para pedir fisiologicamente questões relevantes na rede e níveis celulares levando a uma melhor compreensão da função cerebral e disfunção.

Vídeo: How to Culture, Record and Stimulate Neuronal Networks on Micro-electrode Arrays MEAs

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

A Neuronal e astrocitária Ensaio Co-Cultura de Análise de Conteúdo de Alta de neurotoxicidade

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Biologia

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study angiogenesis but also to study tumorigenesis. After the chick chorioallantoic membrane (CAM) has developed, its blood vessel network can be easily accessed, manipulated and observed and therefore provides an optimal setting for angiogenesis assays. Since the lymphoid system is not fully developed until late stages of incubation, the chick embryo serves as a naturally immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition to nurturing developing allo- and xenografts, the CAM blood vessel network provides a uniquely supportive environment for tumor cell intravasation, dissemination, and vascular arrest and a repository where arrested cells extravasate to form micro metastatic foci.
For experimental purposes, in most of the recent studies the CAM was exposed by cutting a window through the egg shell and experiments were carried out in ovo, resulting in significant limitations in the accessibility of the CAM and possibilities for observation and photo documentation of effects. When shell-less cultures of the chick embryo were used1-4, no experimental details were provided and, if published at all, the survival rates of these cultures were low. We refined the method of ex ovo culture of chick embryos significantly by introducing a rationally controlled extrusion of the egg content. These ex ovo cultures enhance the accessibility of the CAM and chick embryo, enabling easy in vivo documentation of effects and facilitating experimental manipulation of the embryo. This allows the successful application to a large number of scientific questions: (1) As an improved angiogenesis assay5,6, (2) an experimental set up for facilitated injections in the vitreous of the chick embryo eye7-9, (3) as a test environment for dissemination and intravasation of dispersed tumor cells from established cell lines inoculated on the CAM10-12, (4) as an improved sustaining system for successful transplantation and culture of limb buds of chicken and mice13 as well as (5) for grafting, propagation, and re-grafting of solid primary tumor tissue obtained from biopsies on the surface of the CAM14.
In this video article we describe the establishment of a refined chick ex ovo culture and CAM assay with survival rates over 50%. Besides we provide a step by step demonstration of the successful application of the ex ovo culture for a large number of scientific applications. 
Daniel S. Dohle, Susanne D. Pasa, and Sebastian Gustmann contributed equally to this study.

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

The chick chorioallantoic membrane (CAM) is a unique, naturally immunodeficient supportive culture environment to study angiogenesis and tumorigenesis. This video article demonstrates the different steps in chick ex ovo culture, application of potentially angiogenic substances and successful inoculation of tumor cells and tissues on the surface of the CAM.

Pintinho Ex ovo Cultura e Ex ovo CAM Ensaio: como ele realmente funciona

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study ...

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Phenotypes are determined by a complex series of physical (e.g. protein-protein) and functional (e.g. gene-gene or genetic) interactions (GI)1. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7, but GI information remains sparse for prokaryotes8, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10.
Here, we present the key steps required to perform quantitative E. coli Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format. Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g. the 'Keio' collection11) and essential gene hypomorphic mutations (i.e. alleles conferring reduced protein expression, stability, or activity9, 12, 13) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e. slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2 as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9.

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

Mapeamento bacterianas redes funcionais e vias<em&gt; Escherichia Coli</em&gt; Usando sintéticos matrizes genéticas

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

Design and Use of Multiplexed Chemostat Arrays

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is constrained by limiting specific nutrients.1,2 Data from chemostats are highly reproducible for the measurement of quantitative phenotypes as they provide a constant growth rate and environment at steady state. For these reasons, chemostats have become useful tools for fine-scale characterization of physiology through analysis of gene expression3-6 and other characteristics of cultures at steady-state equilibrium.7 Long-term experiments in chemostats can highlight specific trajectories that microbial populations adopt during adaptive evolution in a controlled environment. In fact, chemostats have been used for experimental evolution since their invention.8 A common result in evolution experiments is for each biological replicate to acquire a unique repertoire of mutations.9-13 This diversity suggests that there is much left to be discovered by performing evolution experiments with far greater throughput. 
We present here the design and operation of a relatively simple, low cost array of miniature chemostats&mdash;or ministats&mdash;and validate their use in determination of physiology and in evolution experiments with yeast. This approach entails growth of tens of chemostats run off a single multiplexed peristaltic pump. The cultures are maintained at a 20 ml working volume, which is practical for a variety of applications. It is our hope that increasing throughput, decreasing expense, and providing detailed building and operation instructions may also motivate research and industrial application of this design as a general platform for functionally characterizing large numbers of strains, species, and growth parameters, as well as genetic or drug libraries.

We developed and validated a small-footprint array of miniature chemostats built from readily available parts for low cost. Physiological and experimental evolution results were similar to larger volume chemostats. The ministat array provides a compact, inexpensive, and accessible platform for traditional chemostat experiments, functional genomics, and chemical screening applications.

Concepção e utilização de matrizes quemostato Multiplexados

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is ...

Using an Automated 3D-tracking System to Record Individual and Shoals of Adult Zebrafish

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The presented automatic video tracking system accomplishes this by using a mirror system and a calibration procedure that corrects for the considerable error introduced by the transition of light from water to air. With this system it is possible to record both single and groups of adult zebrafish. Before use, the system has to be calibrated. The system consists of three modules: Recording, Path Reconstruction, and Data Processing. The step-by-step protocols for calibration and using the three modules are presented. Depending on the experimental setup, the system can be used for testing neophobia, white aversion, social cohesion, motor impairments, novel object exploration etc. It is especially promising as a first-step tool to study the effects of drugs or mutations on basic behavioral patterns. The system provides information about vertical and horizontal distribution of the zebrafish, about the xyz-components of kinematic parameters (such as locomotion, velocity, acceleration, and turning angle) and it provides the data necessary to calculate parameters for social cohesions when testing shoals.

The use of a 3D automatic video system that can track individual and groups of zebrafish is described. As application example we explore the effects of the NMDA-receptor antagonist MK-801 on shoals of zebrafish.

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The ...

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein &#945;-subunit) is described in detail.

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

Reação selecionada Monitoramento Espectrometria de Massa para Absolute Quantificação de Proteína

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

Medição e Análise de Produção de Ácido Extracelular determinar Glicosidases Classificação

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

Techniques to Induce and Quantify Cellular Senescence

In response to cellular stress or damage, proliferating cells can induce a specific program that initiates a state of long-term cell-cycle arrest, termed cellular senescence. Accumulation of senescent cells occurs with organismal aging and through continual culturing in vitro. Senescent cells influence many biological processes, including embryonic development, tissue repair and regeneration, tumor suppression, and aging. Hallmarks of senescent cells include, but are not limited to, increased senescence-associated &#946;-galactosidase activity (SA-&#946;-gal); p16INK4A, p53, and p21 levels; higher levels of DNA damage, including &#947;-H2AX; the formation of Senescence-associated Heterochromatin Foci (SAHF); and the acquisition of a Senescence-associated Secretory Phenotype (SASP), a phenomenon characterized by the secretion of a number of pro-inflammatory cytokines and signaling molecules. Here, we describe protocols for both replicative and DNA damage-induced senescence in cultured cells. In addition, we highlight techniques to monitor the senescent phenotype using several senescence-associated markers, including SA-&#946;-gal, &#947;-H2AX and SAHF staining, and to quantify protein and mRNA levels of cell cycle regulators and SASP factors. These methods can be applied to the assessment of senescence in various models and tissues.

Cellular senescence, the irreversible state of cell-cycle arrest, can be induced by various cellular stresses. Here, we describe protocols to induce cellular senescence and methods to assess markers of senescence.

Técnicas para induzir e quantificar a senescência celular

In response to cellular stress or damage, proliferating cells can induce a specific program that initiates a state of long-term cell-cycle arrest, termed ...

An Adipocyte Cell Culture Model to Study the Impact of Protein and Micro-RNA Modulation on Adipocyte Function

Alteration of adipocyte function contributes to the pathogenesis of metabolic diseases including Type 2 diabetes and insulin resistance. This highlights the need to better understand the molecular mechanism involved in adipocyte dysfunction to develop new therapies against obesity-related diseases. Modulating the expression of proteins and micro-RNAs in adipocytes remains highly challenging. This paper describes a protocol to differentiate murine fibroblasts into mature adipocytes and to modulate the expression of proteins and micro-RNAs in mature adipocytes through reverse-transfection using small-interfering RNA (siRNA) and micro-RNA mimicking (miR mimic) oligonucleotides. This reverse-transfection protocol involves the incubation of the transfection reagent and the oligonucleotides to form a complex in the cell culture plate to which the mature adipocytes are added. The adipocytes are then allowed to reattach to the adherent plate surface in the presence of the oligonucleotides/transfection reagent complex. Functional analyses such as the study of insulin signaling, glucose uptake, lipogenesis, and lipolysis can be performed on the transfected 3T3-L1 mature adipocytes to study the impact of protein or micro-RNA manipulation on adipocyte function.

Presented here is a protocol to deliver oligonucleotides such as small-interfering RNA (siRNA), micro-RNA mimics (miRs), or anti-micro-RNA (anti-miR) into mature adipocytes to modulate protein and micro-RNA expression.

Um modelo de cultura celular adipócito para estudar o impacto da modulação de proteína e micro-RNA na função adipócito

Alteration of adipocyte function contributes to the pathogenesis of metabolic diseases including Type 2 diabetes and insulin resistance. This highlights ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Isolamento de miócitos murinos atrial e ventricular de alta qualidade para medidas simultâneas de Ca2+ transitórios e corrente de cálcio tipo L

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...

Como gravar, Cultura e estimular redes neuronais em Micro-Arrays eletrodo (MEA)

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