Name: measuring trans-plasma membrane electron transport by c2c12 myotubes
Uploaded: 2018-05-04T15:00:00.000+00:00
Duration: 10 min 27 s
Description: Watch this Scientific Journal Video about Measuring Trans-Plasma Membrane Electron Transport by C2C12 Myotubes at JoVE.com

Gostaríamos de agradecer seu apoio técnico Thomas Bell, Lyn Mattathil, Mark Mannino e Neej Patel. Este trabalho foi financiado pelo prêmio de serviço de saúde pública dos Estados Unidos R15DK102122 do Instituto Nacional de Diabetes e doenças do rim (NIDDK) e digestivo para Jonathan Fisher. O conteúdo do manuscrito é exclusivamente da responsabilidade dos autores e não representa necessariamente a opinião oficial do NIDDK ou o National Institutes of Health.

Nota: Consulte a Figura 1 para uma visão esquemática das principais etapas.
1. ensaio de redução WST-1
<ol><li>Crescem e se diferenciam células aderentes C2C12, usando de procedimentos de cultura de célula padrão7 em uma placa de 96 poços utilizando linhas A-F.<ol>
<li>Use um meio de diferenciação consistindo de modificado águia de Dulbecco médio (DMEM) suplementado com 2% de soro de cavalo, 100 U/mL penicilina e estreptomicina 0,1 mg/mL. Incube a 37 ° C com 5% CO2células.</li>
		<li>Ao monitorar o envolvimento de ascorbato em tPMET, suplemento meios de diferenciação com o ácido ascórbico 100 µM. Permitir que as células incubar em meios de diferenciação para ~ 24-48 h.</li>
	</ol>
</li>
	<li>Prepare as soluções WST-1 e PMS.<ol>
<li>Para fazer um estoque de 10 mM do WST-1, dissolver 0,033 g do WST-1 (fórmula peso (FW): 651.34 g/mol) em 5 mL de fosfato, tampão salino (PBS). Loja a 4 ° C.</li>
		<li>Para fazer um estoque de 5 mM do PMS, dissolver 0,0023 g de PMS (FW: 306.34 g/mol) em 1,5 mL de deionizada H2O (diH2O). Armazenar a-20 ° C e proteger da luz.</li>
	</ol>
</li>
	<li>Adicionar 0,0108 g de glicose para uma concentração final de 5 mM a 11,4 mL de PBS ou HEPES tampão salino (HBS; sal de sódio HEPES 20 mM, 140 mM de NaCl, 5 mM KCl, 2.5 mM de MgSO4, 1mm CaCl2). Adicione 480 µ l de 10mm WST-1 para uma concentração final de 400 µM e 48 µ l de 5mm PMS para uma concentração final de 20 µM.</li>
	<li>Ao monitorar o envolvimento de ascorbato em tPMET, divida a solução em duas alíquotas de 6 mL. Para uma alíquota, adicione 6 µ l de diH2O e para outra alíquota adicionar 6 µ l de 2 kU/mL para uma concentração final de 2 U/mL.</li>
	<li>Ao monitorar o envolvimento de superóxido em tPMET, divida a solução em duas alíquotas de 6 mL. Para uma alíquota, adicionar 55 µ l de tampão de4 de KPO 0,1 M e para a outra alíquota adicionar µ l 55 de 6,5 kU/mL SOD para uma concentração final de 60 U/mL.</li>
	<li>Lave as células com PBS. Aspirar a mídia e adicionar 150 µ l PBS. Em seguida, Aspire a PBS. Nota: O ensaio é feito com células chapeadas, anexadas. Assim, não há nenhum centrifugação ou o uso de tripsina no processo de lavagem.</li>
	<li>Adicione 100 µ l de solução WST-1 (-) SOD ou (-) de colunas 1-6 e adicionar 100 µ l de solução WST-1 (+) SOD ou (+) de colunas 7-12 na placa de 96 poços. Usar linhas G e H, como controles de fundo (ou seja, para monitorar a mudança em absorvância no reagente sozinho em poços sem células).</li>
	<li>Medir os valores de absorvância utilizando o espectrofotômetro cada 10min por 1h em 438 nm.</li>
	<li>Após a leitura, aspirar a mídia e lave cada uma com 150 µ l de PBS. Em seguida, Aspire a PBS.</li>
	<li>Calcule a variação de absorvância para cada poço subtraindo-se a absorbância inicial para um poco de absorvância em qualquer ponto do tempo para aquele poço. Corrigi essa alteração para a mudança de absorbância (se houver) observada em poços de fundo (ou seja, poços com solução de ensaio, mas sem células).</li>
	<li>Para análise, normalizar os dados de absorbância para a medição de controle de 60 min ou lavar os poços com PBS ou HBS e então executar um ensaio de proteína bicinchoninic ácido (BCA).</li>
	<li>Adicionar o padrão de albumina de soro bovino (BSA) 2 mg/mL (variam de 0,5-2 µ l para a curva padrão, dependendo do grau de confluência das células) aos poços vazios (linhas G e H), em seguida, adicionar o reagente BCA a todos os poços.</li>
	<li>Quantificar os dados via nmol de redução WST-1 por µ g de proteína. Use 37 mM-1 cm-1 22 como o coeficiente de extinção para WST-1 reduzido a 438 nm.</li>
</ol>2. ensaio de redução DPIP
<ol><li>Crescem e se diferenciam células aderentes de C2C12 com o mesmo procedimento como passo 1.1.</li>
	<li>Prepare a solução DPIP de 10mm estoque como segue. Dissolver 0,029 g de DPIP (FW: 290.08 g/mol) em 10 mL de diH2Confirm O. a concentração de DPIP medindo a absorbância em 600 nm com um espectrofotômetro. Use 1 mM-1 cm-1 23 como o coeficiente de extinção para DPIP reduzido em 600 nm. Loja a 4 ° C.</li>
	<li>Adicione 0,0108 g de glicose a 11,880 mL de PBS para uma concentração final de 5 mM. Adicione 120 µ l de 10mm DPIP para uma concentração final de 100 µM.</li>
	<li>Ao monitorar o envolvimento de ascorbato em tPMET, divida a solução em duas alíquotas de 6 mL. Para uma alíquota, adicione 6 µ l de diH2O e para outra alíquota adicionar 6 µ l de 2 kU/mL para uma concentração final de 2 U/mL.</li>
	<li>Ao monitorar o envolvimento de superóxido em tPMET, divida a solução em duas alíquotas de 6 mL. Para uma alíquota, adicionar 55 µ l de tampão de4 de KPO 0,1 M e para a outra alíquota adicionar µ l 55 de 6,5 kU/mL SOD para uma concentração final de 60 U/mL.</li>
	<li>Aspirar a mídia e lavar as células em 150 µ l de PBS. Aspire a PBS e adicionar 100 µ l de solução DPIP (-) SOD ou (-) de colunas 1-6 e adicionar 100 µ l de solução DPIP (+) SOD ou (+) de colunas 7-12 na placa de 96 poços. Usar linhas G e H vontade como controles de fundo (ou seja, para monitorar a mudança em absorvância no reagente sozinho em poços sem células).</li>
	<li>Medir a absorvância a 600 nm utilizando espectrofotômetro cada 10 min para 1 h. quantificar a mudança de absorvância em relação ao controle em semelhante etapas 1.9-1.13 60 min.</li>
</ol>3. a determinação se os aceitadores de electrões reduzida são substratos para AO ou SOD
<ol><li>A 5,436 mL de PBS ou HBS, adicione 240 µ l de 10mm WST-1 para uma concentração final de 400 µM e 24 µ l de 5mm PMS para uma concentração final de 20 µM.<ol>
<li>Para DPIP, adicione 60 µ l de 10mm DPIP, para uma concentração final de 100 µM, a 5,940 mL de PBS ou HBS.</li>
	</ol>
</li>
	<li>Adicione 100 µ l de solução para cada um bem em uma placa de 96 poços de fundo plano na ausência de células e medir a absorvância em espectrofotómetro a 438 nm, para WST-1, ou 600 nm, para DPIP.</li>
	<li>Adicione 1 µ l de ascorbato de 10mm a metade dos poços para uma concentração final de 100 μM. Monitore a absorvância até estabiliza.</li>
	<li>Após estabilização, adicionar 1 µ l de 200 U/mL para uma concentração final de 2 U/mL para cada poço ou adicionar 1 µ l de kU/6ml SOD para uma concentração final de 60 U/mL para cada bem e monitorar a absorvância por 1h.</li>
</ol>

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G., Babcock, G. T., Dye, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+5-methylphenazinium+methyl+sulfate.+Reaction+of+the+oxidized+form+with+NADH+and+of+the+reduced+form+with+oxygen.">Properties of 5-methylphenazinium methyl sulfate. Reaction of the oxidized form with NADH and of the reduced form with oxygen.</a> J Biol Chem. 257 (3), 1458-1461 (1982).</li><li>Maghzal, G. J., Krause, K. H., Stocker, R., Jaquet, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+reactive+oxygen+species+derived+from+the+family+of+NOX+NADPH+oxidases.">Detection of reactive oxygen species derived from the family of NOX NADPH oxidases.</a> Free Radic Biol Med. 53 (10), 1903-1918 (2012).</li></ol>

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Apresentamos dois métodos para a utilização de aceitadores de electrões extracelular, WST-1 e DPIP, em ensaios espectrofotométricos para monitorar tPMET em C2C12 myotubes. Com o crescimento de linhagens celulares em procedimentos padrão de cultura e um leitor de placa de espectrofotômetro, é possível monitorar tPMET com estes aceitadores de electrões em um ensaio de microplacas simples. WST-1 redução é reprodutível de para-bem dentro de um ensaio, mas não há variabilidade do dia a dia. O dia-a-coeficiente...

A visão de que a membrana plasmática tem uma capacidade inerente redox1levou a capacidade das membranas de plasma purificadas de reduzir aceitadores de electrões. Visto anteriormente em fungos, plantas e animais, o tPMET é um processo comum a vários organismos2,3,4,5. Especificamente, este processo tem sido demonstrado em Saccharomyces cerevisiae, cenoura células, hemácias, linfócitos, osteossarcoma, melanoma, macrófagos, músculo esquelético e neutrófilos2,3, 4 , 5 , 6 , 7. em um processo que transporta elétrons através da membrana de plasma para reduzir oxidantes extracelulares, tPMET está envolvido em muitas funções celulares, incluindo: célula crescimento5,8, de viabilidade celular9, ferro metabolismo10, sinalização11,12,13e proteção de1514,12,do espécies reactivas de oxigénio celular. Devido ao envolvimento do tPMET em muitas funções celulares, um desequilíbrio de tPMET tem sido hypothesized para contribuir para o desenvolvimento de algumas condições graves de saúde, incluindo câncer16, doença cardiovascular,17e metabólica Síndrome de18.
Existem várias maneiras de monitorar a transferência de electrões através da membrana de plasma, mas a técnica mais utilizada é avaliar a redução de aceitadores de electrões extracelular através de ensaios colorimétricos. Aceitadores de electrões extracelular comumente usados são sais de tetrazólio, DPIP, BECN e ferricytochrome c19,20. O sal de tetrazólio mais comumente usado é um conhecido sal da segunda geração como WST-119. Este composto é mais fácil de utilizar em ensaios colorimétricos comparados com sais de tetrazólio primeira geração devido a dois grupos sulfonato, que aumentam sua solubilidade de água21. WST-1, em conjunto com o methosulfate de 1-metoxi-Fenazina aceitador elétrons intermediários (mPMS), é reduzido em eventos de transferência de elétron dois. Esta redução altera a forma oxidada fraca-colorido do WST-1 para um mais intenso, amarelo formazan20,22. WST-1 possui um coeficiente de extinção molar elevada de 37 x 103 M-1cm-1, levando a um ensaio de alta sensibilidade21,22. DPIP é também utilizado como um aceitador do elétron extracelular para monitorar tPMET. Tem sido demonstrado que DPIP pode ser diminuído extracelularmente tPMET sem o auxílio de aceitadores de electrões intermediário23,24. Devido à falta de aceitadores de electrões intermediário, DPIP pode diretamente a pick-up os elétrons da membrana plasmática, ao contrário do WST-124. Semelhante a DPIP, BECN foi mostrado para ser diminuído extracelularmente para ferrocianeto de tPMET sem o auxílio de aceitadores de electrões intermediário19,24. Ao contrário do WST-1 e DPIP, BECN tem um coeficiente de extinção molar baixa, levando a uma baixa de sensibilidade de ensaio9. Outra aceitador de electrões extracelular comumente usados para monitorar tPMET é c ferricytochrome semelhante a WST-1, ferricytochrome c redução aumenta com o uso do aceptor de elétrons intermediários, mPMS22. Ao contrário do WST-1, o método de ferricytochrome c é menos sensível devido a um fundo elevado e um coeficiente de extinção molar baixa22.
Aqui nós apresentamos um método de análise em tempo real de tPMET através de ensaios espectrofotométricos. O método utilizado os aceitadores de electrões extracelular WST-1 e DPIP, que tenham um coeficiente de extinção molar alta enquanto ser menos caro em comparação com o outro comumente usado aceitadores de electrões extracelular como ferricytochrome c. Utilizamos o methosulfate Fenazina (PMS) em vez de mPMS têm uma composição química semelhante e PMS é muito menos dispendioso. mPMS fotoquimicamente estável que é uma característica importante para um kit comercial que tem uma vida útil longa. No entanto, fazemos PMS fresco para cada ensaio, por estabilidade não deve ser um problema. Também apresentamos um método para avaliar possíveis interações enzimáticas (ver Figura 1) entre o aceitador de electrões extracelular e enzimas que podem ser utilizadas para caracterizar ainda mais o processo de tPMET. Especificamente, as enzimas AO e SOD podem ser usado determinam qual parte do tPMET é devido ao transporte de ascorbato ou liberação de superóxido extracelular, dois métodos comuns para elétrons ser transportado através da membrana plasmática.

<table><tbody><tr><td>C2C12 myoblasts</td><td>American Type Culture Collection&amp;nbsp;</td><td>CRL-1772</td><td></td></tr><tr><td>Dulbecco&#039;s modified eagle&#039;s medium - low glucose</td><td>Sigma</td><td>D6046</td><td></td></tr><tr><td>Fetal Plex animal serum complex</td><td>Gemini Bio-Products&amp;nbsp;</td><td>100-602</td><td></td></tr><tr><td>penicillin-streptomycin</td><td>Sigma</td><td>516106</td><td></td></tr><tr><td>horse serum</td><td>Gibco Technologies</td><td>16050-130</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate buffered saline</td><td>Sigma</td><td>D8537</td><td></td></tr><tr><td>trypsin-EDTA</td><td>Sigma</td><td>T4049</td><td></td></tr><tr><td>15 cm culture dishes</td><td>TPP</td><td>93150</td><td></td></tr><tr><td>96 well culture plates</td><td>TPP</td><td>92096</td><td></td></tr><tr><td>2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium Sodium Salt (WST-1)</td><td>Accela ChemBio&amp;nbsp; Inc</td><td>SY016315</td><td></td></tr><tr><td>phenazine methosulfate&amp;nbsp;</td><td>Sigma</td><td>P9625</td><td></td></tr><tr><td>L-ascorbic acid</td><td>Sigma</td><td>A5960</td><td></td></tr><tr><td>ascorbate oxidase&amp;nbsp;</td><td>Sigma</td><td>A0157</td><td></td></tr><tr><td>superoxide dismutase&amp;nbsp;</td><td>Sigma</td><td>S5395</td><td></td></tr><tr><td>2,6-dichloroindophenol sodium salt&amp;nbsp;</td><td>ICN Biomedicals</td><td>215011825</td><td></td></tr><tr><td>D-(+)-glucose</td><td>Sigma</td><td>G7528</td><td></td></tr><tr><td>HEPES sodium salt</td><td>Sigma</td><td>H3784</td><td></td></tr><tr><td>sodium chloride</td><td>Sigma</td><td>S7653</td><td></td></tr><tr><td>potassium chloride</td><td>Fisher Scientific&amp;nbsp;</td><td>BP366</td><td></td></tr><tr><td>magnesium sulfate heptahydrate</td><td>Sigma</td><td>M5921</td><td></td></tr><tr><td>calcium chloride dihydrate</td><td>Sigma</td><td>C7902</td><td></td></tr><tr><td>potassium phosphate</td><td>Fisher Scientific&amp;nbsp;</td><td>BP363</td><td></td></tr><tr><td>Pierce BCA Protein Assay Kit</td><td>Thermo Scientific</td><td>23225</td><td></td></tr><tr><td>Powerwave X-I spectrophotometer</td><td>Biotek Insturments</td><td>discontinued&amp;nbsp;</td><td></td></tr><tr><td>Spectronic Genesys 5 Spectrophotometer</td><td>Thermo Scientific</td><td>336001</td><td></td></tr><tr><td>PureGrade 96-well microplate, F-bottom, clear, untreated, non-sterile</td><td>MidSci</td><td>781602</td><td></td></tr><tr><td>Iron (II) chloride tetrahydrate</td><td>Sigma</td><td>220299</td><td></td></tr><tr><td>Iron (II) sulfate heptahydrate</td><td>Sigma</td><td>215422</td><td></td></tr><tr><td>hypoxanthine</td><td>Sigma</td><td>H9636</td><td></td></tr><tr><td>xanthine oxidase</td><td>Sigma</td><td>X4500</td><td></td></tr><tr><td>Excel</td><td>Microsoft</td><td></td><td></td></tr><tr><td>R Studio</td><td>Rstudio</td><td></td><td>https://www.rstudio.com/products/rstudio/</td></tr><tr><td>KC4</td><td>Biotek Insturments</td><td>discontinued&amp;nbsp;</td><td></td></tr></tbody></table>

measuring trans-plasma membrane electron transport by c2c12 myotubes

Transporte de elétrons da membrana trans-plasma (tPMET) desempenha um papel na proteção das células do stress redutivo intracelular, bem como a proteção contra danos por oxidantes extracelulares. Este processo de transporte de elétrons do redutores intracelulares para o extracelulares oxidantes não é bem definido. Aqui apresentamos os ensaios espectrofotométricos por C2C12 myotubes para monitorar tPMET utilizando os aceitadores de electrões extracelular: sal de tetrazólio solúvel em água-1 (WST-1) e 2,6-dichlorophenolindophenol (DPIP ou DCIP). Através da redução destes aceitadores de electrões, somos capazes de monitorar este processo em uma análise em tempo real. Com a adição de enzimas como ascorbato oxidase (AO) e superóxido dismutase (SOD) para os ensaios, podemos determinar qual parte do tPMET é devido à produção de exportação ou superóxido de ascorbato, respectivamente. Enquanto WST-1 foi mostrado para produzir resultados estáveis com baixo fundo, DPIP era capaz de ser re-oxidado após a adição do AO e SOD, que foi demonstrado com análise espectrofotométrica. Este método demonstra um ensaio espectrofotométrico em tempo real, multi bem, rápido, com vantagens sobre outros métodos usados para monitorar tPMET, tais como o ferricianeto (BECN) e redução de c ferricytochrome.

As estatísticas foram realizadas com ANOVA com medidas repetidas utilizando RStudio software estatístico25. Tamanhos de amostra são indicados nas lendas figura.
Para monitorar tPMET, C2C12 myotubes foram utilizados juntamente com aceitadores de electrões extracelular, WST-1 e DPIP. AO foi usado para determinar qual parte do WST-1 e redução de DPIP foi devido ao efluxo de ascorbato e SOD foi usada pa...

Watch this Scientific Journal Video about Measuring Trans-Plasma Membrane Electron Transport by C2C12 Myotubes at JoVE.com

Measuring Trans-Plasma Membrane Electron Transport by C2C12 Myotubes

Trans-Plasma da membrana, transporte de elétrons por Myotubes de C2C12 de medição

O objetivo do presente protocolo é espectrofotometricamente monitorar o transporte de elétrons da membrana trans-plasma utilizando aceitadores de electrões extracelular e analisar interações enzimáticas que podem ocorrer com estes aceitadores de electrões extracelular.

Trans-plasma membrane electron transport (tPMET) plays a role in protection of cells from intracellular reductive stress as well as protection from damage ...

measuring-trans-plasma-membrane-electron-transport-by-c2c12-myotubes

Research

JoVE Journal

Biochemistry

6.9K visualizações.  Saint Louis University. O objetivo do presente protocolo é espectrofotometricamente monitorar o transporte de elétrons da membrana trans-plasma utilizando aceitadores de electrões extracelular e analisar interações enzimáticas que podem ocorrer com estes aceitadores de electrões extracelular.

Vídeo: Measuring Trans-Plasma Membrane Electron Transport by C2C12 Myotubes

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.

The methods presented provide step-by-step instructions for the performance of a collection of microplate based respirometric assays using isolated mitochondria from minimal quantities of mouse skeletal muscle. These assays are able to measure mechanistic changes/adaptations in mitochondrial oxygen consumption in a commonly used animal model.

Usando mitocôndrias isoladas de quantidades mínimas de Rato Músculo Esquelético para alta taxa de transferência de microplacas respiratórias Medições

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of ...

Biologia do Desenvolvimento

Molecular Imaging to Target Transplanted Muscle Progenitor Cells

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle degeneration1, 2. In patients, the ability of resident muscle satellite cells (SCs) to regenerate damaged myofibers becomes increasingly inefficient4. Therefore, transplantation of muscle progenitor cells (MPCs)/myoblasts from healthy subjects is a promising therapeutic approach to DMD. A major limitation to the use of stem cell therapy, however, is a lack of reliable imaging technologies for long-term monitoring of implanted cells, and for evaluating its effectiveness. Here, we describe a non-invasive, real-time approach to evaluate the success of myoblast transplantation. This method takes advantage of a unified fusion reporter gene composed of genes (firefly luciferase [fluc], monomeric red fluorescent protein [mrfp] and sr39 thymidine kinase [sr39tk]) whose expression can be imaged with different imaging modalities9, 10. A variety of imaging modalities, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, and high frequency 3D-ultrasound are now available, each with unique advantages and limitations11. Bioluminescence imaging (BLI) studies, for example, have the advantage of being relatively low cost and high-throughput. It is for this reason that, in this study, we make use of the firefly luciferase (fluc) reporter gene sequence contained within the fusion gene and bioluminescence imaging (BLI) for the short-term localization of viable C2C12 myoblasts following implantation into a mouse model of DMD (muscular dystrophy on the X chromosome [mdx] mouse)12-14. Importantly, BLI provides us with a means to examine the kinetics of labeled MPCs post-implantation, and will be useful to track cells repeatedly over time and following migration. Our reporter gene approach further allows us to merge multiple imaging modalities in a single living subject; given the tomographic nature, fine spatial resolution and ability to scale up to larger animals and humans10,11, PET will form the basis of future work that we suggest may facilitate rapid translation of methods developed in cells to preclinical models and to clinical applications.

A non-invasive means to evaluate the success of myoblast transplantation is described. The method takes advantage of a unified fusion reporter gene composed of genes whose expression can be imaged with different imaging modalities. Here, we make use of a fluc reporter gene sequence to target cells via bioluminescence imaging.

Imagem Molecular para atingir as células progenitoras transplantadas musculares

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle ...

Medicina

Measuring Mitochondrial Electron Transfer Complexes in Previously Frozen Cardiac Tissue from the Offspring of Sow: A Model to Assess Exercise-Induced Mitochondrial Bioenergetics Changes

The mitochondrial electron transfer complex (ETC) profile is modified in the heart tissue of the offspring born to an exercised sow. The hypothesis proposed and tested was that a regular maternal exercise of a sow during pregnancy would increase the mitochondrial efficiency of offspring heart bioenergetics. This hypothesis was tested by isolating mitochondria using a mild-isolation procedure to assess mitochondrial ETC and supercomplex profiles. The procedure described here allowed for the processing of previously frozen archived heart tissues and eliminated the necessity of fresh mitochondria preparation for the assessment of mitochondrial ETC complexes, supercomplexes, and ETC complex activity profiles. This protocol describes the optimal ETC protein complex measurement in multiplexed antibody-based immunoblotting and super complex assessment using blue-native gel electrophoresis.

Preparation of mitochondria-enriched samples from previously frozen archived solid tissues allowed the investigators to perform both functional and analytical assessments of mitochondria in various experimental modalities. This study demonstrates how to prepare mitochondria-enriched preparations from frozen heart tissue and perform analytical assessments of mitochondria.

Medindo complexos de transferência de elétrons mitocondrial em tecido cardíaco previamente congelado da prole da porca: um modelo para avaliar alterações bioenergetics mitocondrial induzidas pelo exercício

The mitochondrial electron transfer complex (ETC) profile is modified in the heart tissue of the offspring born to an exercised sow. The hypothesis ...

Bioquímica

Inner Mitochondrial Membrane Sensitivity to Na+ Reveals Partially Segmented Functional CoQ Pools

Ubiquinone (CoQ) pools in the inner mitochondrial membrane (IMM) are partially segmented to either complex I or FAD-dependent enzymes. Such subdivision can be easily assessed by a comparative assay using NADH or succinate as electron donors in frozen-thawed mitochondria, in which cytochrome c (cyt c) reduction is measured. The assay relies on the effect of Na+ on the IMM, decreasing its fluidity. Here, we present a protocol to measure NADH-cyt c oxidoreductase activity and succinate-cyt c oxidoreductase activities in the presence of NaCl or KCl. The reactions, which rely on the mixture of reagents in a cuvette in a stepwise manner, are measured spectrophotometrically during 4 min in the presence of Na+ or K+. The same mixture is performed in parallel in the presence of the specific enzyme inhibitors in order to subtract the unspecific change in absorbance. NADH-cyt c oxidoreductase activity does not decrease in the presence of any of these cations. However, succinate-cyt c oxidoreductase activity decreases in the presence of NaCl. This simple experiment highlights: 1) the effect of Na+ in decreasing IMM fluidity and CoQ transfer; 2) that supercomplex I+III2 protects ubiquinone (CoQ) transfer from being affected by lowering IMM fluidity; 3) that CoQ transfer between CI and CIII is functionally different from CoQ transfer between CII and CIII. These facts support the existence of functionally differentiated CoQ pools in the IMM and show that they can be regulated by the changing Na+ environment of mitochondria.

Inner Mitochondrial Membrane Sensitivity to Na+ Reveals Partially Segmented Functional CoQ Pools

This protocol describes a comparative assay, using mitochondrial complex activities CI+CIII and CII+CIII in the presence or absence of Na+, to study the existence of partially segmented functional CoQ pools.

Sensibilidade da membrana mitocondrial interna para na+ revela piscinas de COQ funcionais parcialmente segmentadas

Ubiquinone (CoQ) pools in the inner mitochondrial membrane (IMM) are partially segmented to either complex I or FAD-dependent enzymes. Such subdivision ...

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

Preparação e Avaliação respirométrico de mitocôndrias isoladas de tecido muscular esquelético Obtido por meio de agulha de biópsia percutânea

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

Bioenergetic Profile Experiment using C2C12 Myoblast Cells

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in understanding the role of mitochondrial function in obesity, diabetes, aging, cancer, cardiovascular function and safety toxicity.
Cellular metabolism is the process of substrate uptake, such as oxygen, glucose, fatty acids, and glutamine, and subsequent energy conversion through a series of enzymatically controlled oxidation and reduction reactions. These intracellular biochemical reactions result in the production of ATP, the release of heat and chemical byproducts, such as lactate and CO2 into the extracellular environment.
Valuable insight into the physiological state of cells, and the alteration of the state of those cells, can be gained through measuring the rate of oxygen consumed by the cells, an indicator of mitochondrial respiration - the Oxygen Consumption Rate - or OCR. Cells also generate ATP through glycolysis, i.e.: the conversion of glucose to lactate, independent of oxygen. In cultured wells, lactate is the primary source of protons. Measuring the lactic acid produced indirectly via protons released into the extracellular medium surrounding the cells, which causes acidification of the medium provides the Extra-Cellular Acidification Rate - or ECAR.
In this experiment, C2C12 myoblast cells are seeded at a given density in Seahorse cell culture plates. The basal oxygen consumption (OCR) and extracellular acidification (ECAR) rates are measured to establish baseline rates. The cells are then metabolically perturbed by three additions of different compounds (in succession) that shift the bioenergetic profile of the cell.
This assay is derived from a classic experiment to assess mitochondria and serves as a framework with which to build more complex experiments aimed at understanding both physiologic and pathophysiologic function of mitochondria and to predict the ability of cells to respond to stress and/or insults.

A description of a method for profiling mitochondrial function in cells is provided. The mitochondrial profile generated provides four parameters of mitochondrial function that can be measured in one experiment: basal respiration rate, ATP-linked respiration, proton leak, and reserve capacity.

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 ...

Biologia

Modeling Myotonic Dystrophy 1 in C2C12 Myoblast Cells

Myotonic dystrophy 1 (DM1) is a common form of muscular dystrophy. Although several animal models have been established for DM1, myoblast cell models are still important because they offer an efficient cellular alternative for studying cellular and molecular events. Though C2C12 myoblast cells have been widely used to study myogenesis, resistance to gene transfection, or viral transduction, hinders research in C2C12 cells. Here, we describe an optimized protocol that includes daily maintenance, transfection and transduction procedures to introduce genes into C2C12 myoblasts and the induction of myocyte differentiation. Collectively, these procedures enable best transfection/transduction efficiencies, as well as consistent differentiation outcomes. The protocol described in establishing DM1 myoblast cell models would benefit the study of myotonic dystrophy, as well as other muscular diseases.

In this protocol, we present the procedures in establishing myotonic dystrophy 1 myoblast models, including optimized C2C12 cell maintenance, gene transfection/transduction, and myocyte differentiation.

Modelando Myotonic distrofia 1 em células C2C12 de mioblastos

Myotonic dystrophy 1 (DM1) is a common form of muscular dystrophy. Although several animal models have been established for DM1, myoblast cell models are ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Fluorescência baseada em Medição da loja operada entrada de cálcio em células vivas: a partir de células cancerosas cultivadas em fibra muscular esquelética

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

Parâmetro Multi-Medição da abertura dos poros transição de permeabilidade em mitocôndrias isoladas de coração Rato

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

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 ...