Neste experimento, as células C2C12 mioblastos são semeados a uma densidade dada em placas de cultura de células Seahorse. O consumo de oxigênio basal (OCR) e extracelular acidificação (ECAR) taxas são medidas para determinar as taxas de referência. 1. Injeção de células As células são metabolicamente perturbado por três adições de compostos diferentes (em sucessão), que mudar o perfil da bioenergética celular. Um grupo servirá de controle, com execução de mídia adicionado como controle de &quot;compostos&quot;. <ol><li> A primeira injeção é oligomicina. Oligomicina inibe a síntese de ATP através do bloqueio do canal de prótons da porção F o ATP sintase (V Complex). Na pesquisa mitocondrial, que é usado para prevenir o estado 3 (fosforilar) respiração. Com células, ele pode ser usado para distinguir o percentual de consumo de O2 dedicado à síntese de ATP eo percentual de consumo de O2 necessária para superar o vazamento de prótons naturais através da membrana mitocondrial interna. </li><li> A segunda injeção é FCCP. FCCP (carbonil cianeto-p-Trifluormetoxifenilidrazona) é um ionóforo que é um transportador de iões móveis. É um agente desacoplamento porque rompe com a síntese de ATP, transportando íons de hidrogênio através da membrana mitocondrial, em vez do canal de prótons da ATP sintase (V Complex). Este colapso do potencial de membrana mitocondrial leva a um rápido consumo de energia e oxigênio, sem a geração de ATP. Neste caso, tanto OCR e ECAR irá aumentar, devido ao desacoplamento do OCR, e ECAR como as células tentativa de manter seu balanço energético por meio da glicólise para gerar ATP. FCCP tratamento pode ser usado para calcular a capacidade respiratória &quot;reposição&quot; de células que é definida como a diferença quantitativa entre máxima OCR descontrolada eo OCR inicial basal. Tem sido proposto que a manutenção de alguma capacidade respiratória, mesmo em condições de estímulo fisiológico ou fisiopatológico máxima é um fator importante que define a vitalidade e / ou sobrevivência de células. A capacidade das células de responder ao estresse em condições de crescente demanda de energia é em grande parte influenciado pela capacidade bioenergética de mitocôndrias. Esta capacidade bioenergética é determinada por vários fatores, incluindo a capacidade da célula para fornecer substrato para a mitocôndria e da capacidade funcional de enzimas envolvidas no transporte de elétrons </li><li> Na terceira injecção, rotenona, um inibidor do Complexo I, é adicionada às células. Isto irá desligar a respiração mitocondrial e permitir que tanto as frações mitocondrial e não contribuindo para a respiração mitocondrial deve ser calculado. Observa-se uma diminuição no OCR, devido à função mitocondrial danificada, com um aumento concomitante ECAR como a célula passa para um estado mais glicolítica, a fim de manter seu balanço energético Rotenona é um inibidor mitocondrial que impede a transferência de elétrons do centro Fe-S no Complexo I ubiquinona (coenzima Q). Esta inibição do Complexo I impede a energia potencial em NADH de ser convertida em energia utilizável na forma de ATP. </li></ol> 2. Reagentes e materiais <ol><li> Oligomicina, FCCP, e Soluções de rotenona (Seahorse Mito Kit Stress Test) </li><li> DMEM Executando Media (Seahorse # 100965-000) </li><li> DMSO (Sigma D8418) </li><li> Água destilada (Gibco 15230-170) </li><li> Tampão de calibração (Seahorse Bioscience) </li></ol> 3. Meio de Crescimento <ol><li> 500 mL DMEM (Gibco 11965-092) </li><li> FBS 10% (Hyclone SH90070.03) </li><li> 5 mL Penn / Strep (Gibco 15140-122) </li><li> 5 mL piruvato de sódio (Sigma S8636) </li><li> 5 mL Glutamax (Gibco 35050-061) </li></ol> 4. Semeadura Protocolo <ol><li> Células são semeadas em culturas de células XF96 placas com 10.000 células / poço em 100 mL de meio de crescimento e colocou em 37 ° C incubadora com 10% de CO 2. </li><li> Células irão aderir à placa de cultura de células XF96 dentro de 1 hora. </li><li> Ensaio em células XF96 24 horas após a semeadura. </li></ol> 5. Elaboração de modelos de Ensaio <ol><li> Usando o Assistente de Ensaio (Anexo I), gerar um modelo com o esquema seguinte grupo: <img src="/files/ftp_upload/2511/2511fig1.jpg" alt="Figura 1" /> Figura 1. Layout de grade Bem identificar atribuições coluna e do grupo </li></ol> 6. Preparação composto <ol><li> Prepare os seguintes compostos na XF DMEM Ensaio de mídia da seguinte forma: 10 oligomicina uM, 30,0 uM FCCP, 20,0 mM rotenona, Estas concentrações representam a diluição de 10X que serão feitas quando os compostos são injetados dentro do poço. As concentrações de trabalho são: 1 oligomicina uM, 3,0 uM FCCP, 2,0 mM rotenona </li></ol> 7. Mudança de mídia e preparação celular <ol&gt; <li> Coloque a placa de célula na Prep XF Station </li><li> Definir o volume final de média a 160 mL por poço. </li><li> Incubar 37 ° C em incubadora de CO 2 sem por 60 minutos para permitir que as células pré-equilibrar com o meio de ensaio. </li></ol> 8. Como carregar o cartucho Sensor <ol><li> Compostos quente a 37 ° C antes de carregar cartucho de sensor de carga e os compostos nas portas injector da seguinte forma: <ol><li> Colunas 1-4: Carga 16, 18 e 20 mL de XF Assay media (DMEM) em portos A, B e C, respectivamente. </li><li> Colunas 5-12: 16 mL de carga nos portos A oligomicina 18 mL de carga em portos FCCP B Carga de 20 mL de rotenona em portos C </li></ol></li></ol> 9. Comandos protocolo <table border="1"><tbody><tr><td> Comando </td><td> Tempo (min) </td><td> Porto </td></tr><tr><td> Calibrar </td><td></td><td></td></tr><tr><td> Equilibrar </td><td></td><td></td></tr><tr><td> Loop Start </td><td> 3X </td><td></td></tr><tr><td> Mistura </td><td> 3 </td><td></td></tr><tr><td> Esperar </td><td> 2 </td><td></td></tr><tr><td> Medida </td><td> 3 </td><td></td></tr><tr><td> Loop End </td><td></td><td></td></tr><tr><td> Injetar </td><td></td><td> A </td></tr><tr><td> Loop Start </td><td> 2X </td><td></td></tr><tr><td> Mistura </td><td> 3 </td><td></td></tr><tr><td> Esperar </td><td> 2 </td><td></td></tr><tr><td> Medida </td><td> 3 </td><td></td></tr><tr><td> Injetar </td><td></td><td> B </td></tr><tr><td> Loop Start </td><td> 2X </td><td></td></tr><tr><td> Mistura </td><td> 3 </td><td></td></tr><tr><td> Esperar </td><td> 2 </td><td></td></tr><tr><td> Medida </td><td> 3 </td><td></td></tr><tr><td> Injetar </td><td></td><td> C </td></tr><tr><td> Loop Start </td><td> 2X </td><td></td></tr><tr><td> Mistura </td><td> 3 </td><td></td></tr><tr><td> Esperar </td><td> 2 </td><td></td></tr><tr><td> Medida </td><td> 3 </td><td></td></tr><tr><td> Final </td><td></td><td></td></tr></tbody></table> Comandos da tabela 1. Protocolo

<ol>
	<li>Choi, W. S., Gerencser, A. A., Nicholls, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioenergetic+analysis+of+isolated+cerebrocortical+nerve+terminals+on+a+microgram+scale:+spare+respiratory+capacity+and+stochastic+mitochondrial+failure.">Bioenergetic analysis of isolated cerebrocortical nerve terminals on a microgram scale: spare respiratory capacity and stochastic mitochondrial failure.</a> J Neurochem. 109, 1179-1191 (2009).</li><li>Hill, B. G., Dranka, B. P., Zou, L., Chatham, J. C., Darley-Usmar, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+the+bioenergetic+reserve+capacity+in+response+to+cardiomyocyte+stress+induced+by+4-hydroxynonenal.">Importance of the bioenergetic reserve capacity in response to cardiomyocyte stress induced by 4-hydroxynonenal.</a> Biochem J. 424, 99-107 (2009).</li><li>Liu, J., Cao, L., Chen, J., Song, S., Lee, I. H., Quijano, C., Liu, H., Keyvanfar, K., Chen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bmi1+regulates+mitochondrial+function+and+the+DNA+damage+response+pathway.">Bmi1 regulates mitochondrial function and the DNA damage response pathway.</a> Nature. , 459-7245 (2009).</li><li>Malmgren, S., Nicholls, D. G., Taneera, J., Bacos, K., Koeck, T., Tamaddon, A., Wibom, R., Groop, L., Ling, C., Mulder, H., Sharoyko, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tight+coupling+between+glucose+and+mitochondrial+metabolism+in+clonal+beta-cells+is+required+for+robust+insulin+secretion.">Tight coupling between glucose and mitochondrial metabolism in clonal beta-cells is required for robust insulin secretion.</a> J Biol Chem. 284, 32395-32404 (2009).</li><li>Dranka, B. P., Hill, B. G., Darley-Usmar, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+reserve+capacity+in+endothelial+cells:+The+impact+of+nitric+oxide+and+reactive+oxygen+species.">Mitochondrial reserve capacity in endothelial cells: The impact of nitric oxide and reactive oxygen species.</a> Free Radic Biol Med. 48, 905-914 (2010).</li><li>Perez, J., Hill, B. G., Benavides, G. A., Dranka, B. P., Darley-Usmar, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+cellular+bioenergetics+in+smooth+muscle+cell+proliferation+induced+by+platelet-derived+growth+factor.">Role of cellular bioenergetics in smooth muscle cell proliferation induced by platelet-derived growth factor.</a> Biochem J. 428, 255-267 (2010).</li><li>Mor&#225;n, M., Rivera, H., S&#225;nchez-Arag&#243;, M., Bl&#225;zquez, A., Merinero, B., Ugalde, C., Arenas, J., Cuezva, J. M., Mart&#237;n, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+bioenergetics+and+dynamics+interplay+in+complex+I-deficient+fibroblasts.">Mitochondrial bioenergetics and dynamics interplay in complex I-deficient fibroblasts.</a> Biochim Biophys Acta. , 1802-185 (2010).</li><li>C&#225;rdenas, C., Miller, R. A., Smith, I., Bui, T., Molg&#243;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+regulation+of+cell+bioenergetics+by+constitutive+InsP3+receptor+Ca2++transfer+to+mitochondria.">Essential regulation of cell bioenergetics by constitutive InsP3 receptor Ca2+ transfer to mitochondria.</a> Cell. , 142-142 (2010).</li></ol>

Min Wu, Per Bo Jensen, W. George Rogers, e David A. Ferrick são funcionários da Seahorse Bioscience, que produz reagentes e instrumentação apresentado neste artigo.

Este ensaio é derivado da experiência clássica para sondar a função mitocondrial e serve como um quadro com o qual construir experiências mais complexas como objetivo compreender várias mudanças no metabolismo celular, a função mitocondrial, e bioenergética global. Todos os compostos utilizados neste experimento devem ser otimizados para a concentração que proporciona o efeito máximo. Ou seja, deve-se realizar experimentos de titulação separado para verificar esses valores. Isto é especialmente importante com FCCP, como a curva de titulação tende a ser bastante acentuada, e FCCP em excesso pode realmente diminuir as respostas em OCR. Intervalos típicos (concentração final) para teste seria: <ul><li> 0,1-1,0 ug / mL de oligomicina </li><li> 0,1-5,0 uM FCCP </li><li> 0,1-1,0 rotenona uM </li></ul> Note-se que as respostas a cada composto acima (especialmente FCCP) será influenciada pela composição do ensaio de mídia (tipo base, [glicose], [piruvato], presença / ausência de BSA, etc). Além disso, se o ensaio XF composição media é alterada, otimização terá de ser re-executadas. A presença e concentração de piruvato é especialmente importante na obtenção da capacidade respiratória máxima, devido à FCCP. Seahorse Bioscience tem observado em um número de linhas de células que omissão de piruvato anula a capacidade das células para responder ao máximo (acima de linha de base) para FCCP. Tipicamente, as concentrações de piruvato mM 10/01 deve ser testado para entender a melhor concentração de piruvato para obter a respiração máxima. Note-se que [piruvato] e [glicose] pode ser necessário &quot;cross-titulada&quot; para obter as condições ideais de mídia para o experimento. Os resultados típicos deste experimento são apresentados a seguir em um gráfico que mostra OCR vs tempo e outro mostrando ECAR versus tempo: <img src="/files/ftp_upload/2511/2511fig2.jpg" alt="Figura 2" /> Figura 2. OCR Tempo vs <img src="/files/ftp_upload/2511/2511fig3.jpg" alt="Figura 3" /> Figura 3. ECAR Tempo vs Aqui observamos as respostas esperadas em OCR e ECAR como as células são tratadas com cada composto sucessivas. Para oligomicina, OCR diminui como resultado do bloqueio da síntese de ATP mitocondrial no Complexo V. Uma vez que as células são incapazes de sintetizar ATP via OXPHOS, voltam a glicólise para atender à demanda de ATP, portanto, observamos um aumento na ECAR. Como mostrado anteriormente, FCCP atua como um agente desacoplamento. Uma vez que as células precisam agora superar o vazamento de prótons através da membrana mitocondrial interna, OCR aumenta significativamente a O2 é consumido mais para bombear o excesso de prótons de volta através da membrana mitocondrial. Finalmente, rotenona inibe mitocondrial Complexo I e Complexo III, respectivamente, o que faz com que o fluxo de elétrons para cessar na cadeia de transporte de elétrons e, assim, o consumo de O2 é reduzido drasticamente. <img src="/files/ftp_upload/2511/2511fig4.jpg" alt="Figura 4" /> Figura 4. Respiração parâmetros Além das alterações esperadas na respiração e ECAR, uma série de parâmetros respiratórios podem ser obtidas a partir destes dados. Isto é resumido na figura acima: Aqui vemos que podemos obter informações sobre a respiração basal das células, o percentual de consumo de O2 dedicada à produção de ATP, bem como o montante atribuído para a manutenção do gradiente de prótons (H + devido ao vazamento). Além disso, podemos obter a taxa respiratórias máximas em condições de respiração desacoplado (por vezes referido como reposição da capacidade respiratória) e, finalmente, podemos determinar a quantidade de consumo de O2 não devido a processos mitocondriais. Um número crescente de estudos estão empregando este perfil mitocondrial para avaliar bioenergética celular, identificar disfunção mitocondrial e para prever a habilidade das células de responder ao estresse e / ou insultos. Para mais informações e detalhes sobre este método experimental ea idéia de capacidade respiratória de reserva, por favor veja se referir as seguintes publicações 1-8.

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bioenergetic profile experiment using c2c12 myoblast cells

A capacidade de medir o metabolismo celular e compreender a disfunção mitocondrial, permitiu que os cientistas em todo o mundo para fazer avançar a sua investigação na compreensão do papel da função mitocondrial em obesidade, diabetes toxicidade, envelhecimento, câncer, função cardiovascular e segurança. Metabolismo celular é o processo de absorção do substrato, tais como oxigênio, glicose, ácidos graxos, e glutamina, e subseqüente conversão de energia através de uma série de oxidação enzimática controlada e as reações de redução. Estas reações bioquímicas intracelulares resultam na produção de ATP, a liberação de calor e produtos químicos derivados, tais como lactato e CO 2 no ambiente extracelular. Informações valiosas sobre o estado fisiológico das células, ea alteração do estado das células, pode ser adquirida através da medição da taxa de oxigênio consumido pelas células, um indicador da respiração mitocondrial - a Taxa de Consumo de Oxigênio - ou OCR. Células também gerar ATP através da glicólise, ou seja: a conversão de glicose em lactato, independente do oxigênio. Em poços de cultura, o lactato é a principal fonte de prótons. Medindo o ácido láctico produzido indiretamente através de prótons lançado no meio extracelular em torno das células, o que provoca a acidificação do meio fornece a taxa de acidificação extra-celular - ou ECAR. Neste experimento, as células C2C12 mioblastos são semeados a uma densidade dada em placas de cultura de células Seahorse. O consumo de oxigênio basal (OCR) e extracelular acidificação (ECAR) taxas são medidas para determinar as taxas de referência. As células são então metabolicamente perturbado por três adições de compostos diferentes (em sucessão), que mudar o perfil da bioenergética celular. Este ensaio é derivado de uma experiência clássica para avaliar a mitocôndria e serve como um quadro com o qual construir experiências mais complexas como objetivo compreender a função tanto fisiológicas e fisiopatológicas das mitocôndrias e para prever a habilidade das células de responder ao estresse e / ou insultos.

Watch this Scientific Journal Video about Bioenergetic Profile Experiment using C2C12 Myoblast Cells at JoVE.com

Bioenergetic Profile Experiment using C2C12 Myoblast Cells

A descrição de um método para caracterização da função mitocondrial em células é fornecido. O perfil mitocondrial gerado fornece quatro parâmetros da função mitocondrial, que pode ser medido em um experimento: taxa de respiração basal, ATP-linked respiração, vazamento de prótons, e capacidade de reserva.

Experimento Perfil bioenergética usando células C2C12 mioblastos

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in ...

bioenergetic-profile-experiment-using-c2c12-myoblast-cells

Research

JoVE Journal

Biology

50.3K visualizações.  Novato, CA. A descrição de um método para caracterização da função mitocondrial em células é fornecido. O perfil mitocondrial gerado fornece quatro parâmetros da função mitocondrial, que pode ser medido em um experimento: taxa de respiração basal, ATP-linked respiração, vazamento de prótons, e capacidade de reserva.

Vídeo: Bioenergetic Profile Experiment using C2C12 Myoblast Cells

Testing Nicotine Tolerance in Aphids Using an Artificial Diet Experiment

Plants may upregulate the production of many different seconday metabolites in response to insect feeding. One of these metabolites, nicotine, is well know to have insecticidal properties. One response of tobacco plants to herbivory, or being gnawed upon by insects, is to increase the production of this neurotoxic alkaloid. Here, we will demonstrate how to set up an experiment to address this question of whether a tobacco-adapted strain of the green peach aphid, Myzus persicae, can tolerate higher levels of nicotine than the a strain of this insect that does not infest tobacco in the field.

In this video and assay is demonstrated that tests the tolerance of nicotine to two types of aphids one that infests tobacco plants in the field and one that does not.

Teste de tolerância à nicotina em Aphids Usando uma experiência Dieta Artificial

Plants may upregulate the production of many different seconday metabolites in response to insect feeding. One of these metabolites, nicotine, is well ...

Biologia

Morris Water Maze Experiment

The Morris water maze is widely used to study spatial memory and learning. Animals are placed in a pool of water that is colored opaque with powdered non-fat milk or non-toxic tempera paint, where they must swim to a hidden escape platform. Because they are in opaque water, the animals cannot see the platform, and cannot rely on scent to find the escape route. Instead, they must rely on external/extra-maze cues. As the animals become more familiar with the task, they are able to find the platform more quickly. Developed by Richard G. Morris in 1984, this paradigm has become one of the "gold standards" of behavioral neuroscience.

The Morris water maze is a well-accepted tool used to document the involvement of the hippocampus in a behavioral task.

Morris Experiment Maze Água

The Morris water maze is widely used to study spatial memory and learning. Animals are placed in a pool of water that is colored opaque with powdered ...

Eukaryotic Polyribosome Profile Analysis

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase or the elongation phase by a variety of cellular stresses such as amino acid starvation or growth factor withdrawal. Alternatively, translation of individual mRNAs can be regulated by mRNA localization or the presence of cognate microRNAs. Studies of protein synthesis frequently utilize polyribosome analysis to shed light on the mechanisms of translation regulation or defects in protein synthesis. In this assay, mRNA/ribosome complexes are isolated from eukaryotic cells.  A sucrose density gradient separates mRNAs bound to multiple ribosomes known as polyribosomes from mRNAs bound to a single ribosome or monosome. Fractionation of the gradients allows isolation and quantification of the different ribosomal populations and their associated mRNAs or proteins. Differences in the ratio of polyribosomes to monosomes under defined conditions can be indicative of defects in either translation initiation or elongation/termination. Examination of the mRNAs present in the polyribosome fractions can reveal whether the cohort of individual mRNAs being translated changes with experimental conditions. In addition, ribosome assembly can be monitored by analysis of the small and large ribosomal subunit peaks which are also separated by the gradient. In this video, we present a method for the preparation of crude ribosomal extracts from yeast cells, separation of the extract by sucrose gradient and interpretation of the results.  This procedure is readily adaptable to mammalian cells.

This article describes a protocol for the extraction of translating ribosomes from eukaryotic cells. Once extracted, ribosomes are separated into monosomes and polyribosomes by sucrose gradient fractionation to allow different ribosomal populations to be analyzed. As such, this method is the gold standard for examining the regulation of translation.

Análise de Perfil eucarióticas Polyribosome

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase ...

Metabolic Profile Analysis of Zebrafish Embryos

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these relationships will help to understand the underlying etiology for a range of diseases linked with mitochondrial dysfunction, such as diabetes and obesity. Recent advances in instrumentation, has enabled the monitoring of distinct parameters of mitochondrial function in cell lines or tissue explants. Here we present a method for a rapid and sensitive analysis of mitochondrial function parameters in vivo during zebrafish embryonic development using the Seahorse bioscience XF 24 extracellular flux analyser. This protocol utilizes the Islet Capture microplates where a single embryo is placed in each well, allowing measurement of bioenergetics, including: (i) basal respiration; (ii) basal mitochondrial respiration (iii) mitochondrial respiration due to ATP turnover; (iv) mitochondrial uncoupled respiration or proton leak and (iv) maximum respiration. Using this approach embryonic zebrafish respiration parameters can be compared between wild type and genetically altered embryos (mutant, gene over-expression or gene knockdown) or those manipulated pharmacologically. It is anticipated that dissemination of this protocol will provide researchers with new tools to analyse the genetic basis of metabolic disorders in vivo in this relevant vertebrate animal model.

Zebrafish represent a powerful vertebrate model that has been under-utilised for metabolic studies. Here we describe a rapid way to measure the in vivo metabolic profile of developing zebrafish that allows the comparison of different mitochondrial function parameters between genetically or pharmacologically manipulated embryos, thereby increasing the applicability of this organism.

Análise do Perfil Metabólico de embriões Zebrafish

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Indução e Análise de epitelial para mesenquimal Transição

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem cells of skeletal muscle, the myogenic precursor cells (MPCs), have proven indispensible to our understanding of mammalian skeletal muscle development and growth. Particularly among the basal taxa of Vertebrata, however, data are limited describing the molecular mechanisms controlling the self-renewal, proliferation, and differentiation of MPCs. Of particular interest are potential mechanisms that underlie the ability of basal vertebrates to undergo considerable postlarval skeletal myofiber hyperplasia (i.e.&#160;teleost fish) and full regeneration following appendage loss (i.e.&#160;urodele amphibians). Additionally, the use of cultured myoblasts could aid in the understanding of regeneration and the recapitulation of the myogenic program and the differences between them. To this end, we describe in detail a robust and efficient protocol (and variations therein) for isolating and maintaining MPCs and their progeny, myoblasts and immature myotubes, in cell culture as a platform for understanding the evolution of the myogenic program, beginning with the more basal vertebrates. Capitalizing on the model organism status of the zebrafish (Danio rerio), we report on the application of this protocol to small fishes of the cyprinid clade Danioninae. In tandem, this protocol can be utilized to realize a broader comparative approach by isolating MPCs from the Mexican axolotl (Ambystomamexicanum) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream1-4.

In vitro culture systems have proven indispensible to our understanding of vertebrate myogenesis. However, much remains to be learned about nonmammalian skeletal muscle development and growth, particularly in basal taxa. An efficient and robust protocol for isolating the adult stem cells of this tissue, the myogenic precursor cells (MPCs), and maintaining their self-renewal, proliferation, and differentiation in a primary culture setting allows for the identification of conserved and divergent regulatory mechanisms throughout the vertebrate lineages.

Preparação de Primário Miogênica de Células Precursoras / Culturas de mioblastos de Vertebrados basais Linhagens

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem ...

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and composed of mature enterocytes, goblet cells and enteroendocrine cells; and crypts, residing proximal to the submucosa and the muscularis, harboring adult stem and progenitor cells and mature Paneth cells, as well as stromal and immune cells of the crypt microenvironment. Until the last few years, in vitro studies of small intestine was limited to cell lines derived from either benign or malignant tumors, and did not represent the physiology of normal intestinal epithelia and the influence of the microenvironment in which they reside. Here, we demonstrate a method adapted from Sato et al. (2009) for culturing primary mouse intestinal crypt organoids derived from C57BL/6 mice. In addition, we present the use of crypt organoid cultures to assay the crypt metabolic profile in real time by measurement of basal oxygen consumption, glycolytic rate, ATP production and respiratory capacity. Organoids maintain properties defined by their source and retain aspects of their metabolic adaptation reflected by oxygen consumption and extracellular acidification rates. Real time metabolic studies in this crypt organoid culture system are a powerful tool to study crypt organoid energy metabolism, and how it can be modulated by nutritional and pharmacological factors.

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

Small intestinal crypt organoids cultured ex vivo provide a tissue culture system that recapitulates growth of crypts dependent on stem cells and their niche. We established a method to assay the metabolic profile in real time in primary mouse crypt organoids. We found organoids maintain physiological properties defined by their source.

Tempo real Análise do Perfil metabólico em<em&gt; Ex Vivo</em&gt; Culturas mouse Intestinal Crypt organóide

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Nefrotoxina microinjeção no peixe-zebra ao modelo de lesão renal aguda

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are the recently discovered modified cytosine bases produced by enzymatic oxidation of 5mC, whose biological functions remain relatively obscure. A number of approaches ranging from biochemical to antibody based techniques have been employed to study the genomic distribution and global content of these modifications in various biological systems. Although some of these approaches can be useful for quantitative assessment of these modified forms of 5mC, most of these methods do not provide any spatial information regarding the distribution of these DNA modifications in different cell types, required for correct understanding of their functional roles. Here we present a highly sensitive method for immunochemical detection of the modified forms of cytosine. This method permits co-detection of these epigenetic marks with protein lineage markers and can be employed to study their nuclear localization, thus, contributing to deciphering their potential biological roles in different experimental contexts.

Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.

Detecção de formas modificadas da citosina Utilizando imunohistoquímica Sensível

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Uma interface de usuário gráfica para rastreamento assistido por software da concentração de proteínas em protrusões celulares dinâmicas

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...