Este trabalho foi financiado pelo Instituto Nacional do Envelhecimento (AG04418), Instituto Nacional de Abuso de Drogas, Instituto Nacional do Programa de Saúde Karolinska Institutet Parcerias Pós-Graduação, Karolinska Institutet, da Suécia de Pesquisa do Conselho, Brain Power Sueco e Fundação Cérebro sueco. Muito obrigado a Mattias Karlen e Giuseppe Dr. Coppotelli de apoio criativo com a Figura 1 e 2, respectivamente; Karin Pernold de assistência técnica; e as Dras. Barry J. Hoffer, Lars Olson, e Nils-Göran Larsson para o conselho útil muito e discussão.

1. Preparação de tecidos para cryosectioning <ol><li> Sacrificar o animal por qualquer deslocamento cervical ou a decapitação, em conformidade com a ética permitir disponíveis. </li><li> Recolher rapidamente os tecidos de interesse (por exemplo. Cérebro), e rapidamente congelar em gelo seco (tecidos podem exigir congelamento em isopentano ou propano refrigerado com nitrogênio líquido para obter a morfologia ideal). Loja de tecidos em folha de alumínio a -80 ° C até que esteja pronto para a seção. </li><li> Incorporar tecidos congelados em preparação para cryosectioning. </li><li> Coletar seções 14 mícrons criostato a -21 ° C (pode ser necessário para ajustar a temperatura ± 02/01 ° C). Seções descongelar em slides usando bloco de aquecimento, e slides loja sem lamínulas a -20 ° C até que esteja pronto para uso. </li></ol> 2. Histoquímica COX <ol><li> Permitir slides para secar em temperatura ambiente por 1 hora. Coloque slides em uma câmara de corrediça mancha com papel de filtro molhado, cortado em tiras. Para obter consistenresultados t em cada experimento, recomenda-se a processo de um máximo de dez slides por experimento para minimizar os atrasos. </li><li> Prepare sob um capuz químico 1X DAB, 100 mM citocromo c em 0,1 M PBS pH = 7,0. Vortex rapidamente. </li><li> Adicionar 2 g de bovinos catalase (2 mg ml -1 ou aproximadamente 4 UI ml -1). Misturar bem em vórtice para quebrar todos os grãos de catalase. </li><li> Aplicar 150-200 mL de meio de incubação para cada slide, use pipeta de espalhar uniformemente em todas as seções. </li><li> Incubar as lâminas por 40 minutos a 37 ° C. </li><li> Remover excesso de solução da slides. Lavagem das lâminas 4 vezes, 10 minutos de cada vez, em 0,1 M PBS pH = 7,0. </li><li> Retornar slides para deslizar coloração de câmara com tiras de papel molhado. </li></ol> 3. Histoquímica SDH <ol><li> Prepare sob um capuz químico 1,5 mM NBT, 130 mM succinato de sódio, 0,2 mM PMS, e 1,0 mM de azida de sódio em 0,1 M PBS pH = 7,0. Tome cuidado para proteger aPMS da luz. Vortex rapidamente. </li><li> Aplicar 150-200 mL de meio de incubação para cada slide, use pipeta de espalhar uniformemente em todas as seções. </li><li> Incubar as lâminas por 40 minutos a 37 ° C. </li><li> Remover excesso de solução da slides. Lavagem das lâminas 4 vezes, 10 minutos de cada vez, em 0,1 M PBS pH = 7,0. </li><li> Desidratar as lâminas durante 2 minutos nas seguintes concentrações de etanol: 70%, 70%, 95%, 95%, 99,5%. Em seguida, permitir que 10 minutos em uma etapa adicional de 99,5%. </li><li> Coloque as lâminas em xilol por 10 minutos. Montar com Entellan e lamínula. Permitir que os slides para secar horas durante a noite, ou pelo menos 1-2 em uma área ventilada. </li></ol> 4. Determinação de disfunção mitocondrial <ol><li> A quantidade de disfunção mitocondrial é indicado pela quantidade de coloração azul celular. Para semi-quantificar estes valores, slides deve ser codificado e visualizados sob microscopia de campo brilhante. Semi-quantificação devem ser realizados em um cego<html> base utilizando uma escala, por exemplo, 0-4 (0, coloração azul não; 4, apenas coloração azul). É melhor realizar esse tipo de semi-quantificação de várias seções de um determinado assunto / animal para calcular um valor médio para cada disciplina / animal. </li><li> Estatísticas devem ser realizados através de um teste não-paramétrico, como Mann-Whitney ou Kruskal-Wallis. </li></ol> 5. Controles adequados especificidade <ol><li> Para controles especificidade para a atividade COX, repita &quot;histoquímica COX&quot; passos e adicionar 2,5 mM de azida de sódio, um inibidor da cadeia respiratória terminal. </li><li> Para controles especificidade para a atividade SDH, repita &quot;histoquímica SDH&quot; passos com a remoção de succinato de sódio ea adição de 50 mM malonato, um inibidor competitivo da SDH. </li><li> Lavar e desidratar seções em uma série de etanol, e depois montar e lamínula os slides conforme descrito nas etapas 3,4-3,6. </li></ol> 6. Resultados representativos: tenda &quot;&gt; O esquema geral dos exemplos COX / SDH duplo rotulagem ensaio histoquímico é ilustrada na Figura 2. Representante da histoquímica COX / SDH duplo rotulagem adequada no cérebro de seções do tipo selvagem e camundongos mutator prematuramente envelhecimento mtDNA são mostrados na Figura 3. A coloração marrom escuro em camundongos selvagens (Figura 3, painel da esquerda) mostrou atividade COX normal. células com deficiências da cadeia respiratória, indicada pela coloração azul, foram revelados em 12 semanas de idade ratos mutator mtDNA, e essas deficiências se tornou mais difundido como ratos mutator mtDNA idade para 46 semanas (Figura 3, centro e painel da direita). Exemplos de inadequado COX / SDH dupla rotulagem em seções do cérebro de camundongos selvagens, devido à rotulagem COX insuficiente são mostrados na Figura 4. Tempo de incubação insuficiente para a demonstração da atividade da COX, ou reduzir a disponibilidade de oxigênio molecular pela cobertura das deslize durante a incubação, resultou em uma redução da deposição de reagir DABíon produto e, assim, permitido para a formação do produto final formazan azul durante a incubação SDH. Atividades COX e SDH também pode ser investigado separadamente (Figura 5, esquerda e centro), no entanto, a rotulagem seqüencial é útil na identificação de células com deficiências COX, devido à formação do precipitado azul durante a incubação SDH (Figura 3, centro e à direita). Controles especificidade para COX atividades e SDH também pode ser feito (Figura 5, direita). <img alt="Figura 1" src="/files/ftp_upload/3266/3266fig1.jpg" /> Figura 1. Respiratória mitocondrial Complexos IV. A cadeia respiratória mitocondrial está localizado dentro da membrana interna e inclui cinco complexos. A finalidade da cadeia respiratória é transportar elétrons do complexo I a IV e ao fazê-lo cria um gradiente de prótons através da membrana interna usada por V Complex (ATPase) para produzir ATP. Represe hexágonos vermelhosubunidades nt codificadas por mtDNA. Hexágonos brancos representam subunidades codificadas pelo DNA nuclear (note que o complexo II é completamente codificada a partir do genoma nuclear). Assim, mutações no genoma mitocondrial pode causar disfunção da cadeia respiratória devido a mutações nas subunidades dos complexos da cadeia respiratória. <img alt="Figura 2" src="/files/ftp_upload/3266/3266fig2.jpg" /> Figura 2. Fluxograma do ensaio COX / SDH duplo rotulagem histoquímica. Dissecar os órgãos de interesse, rapidamente congelar os tecidos em gelo seco, e guarde-a - 80 ° C. Coletar seções criostato e manter a - 20 ° C até usar. Permitir seções para ar seco em temperatura ambiente por 1 hora. Preparar o meio de incubação para histoquímica COX, aplicá-lo à slides, e incubar por 40 minutos a 37 ° C. Lavar as seções em PBS 4 vezes por 10 minutos cada lavagem. Preparar o meio de incubação para histoquímica SDH, aplicá-la ao slides, e novamente incubar por 40 minutos a 37 ° C. Lavar as seções novamente em PBS, desidratar em série etanólica, e depois montar e lamínula os slides. A COX / SDH duplo rotulados seções estão prontos para ver brilhantes sob microscopia de campo dentro de 1-2 horas. <img alt="Figura 3" src="/files/ftp_upload/3266/3266fig3.jpg" /> Figura 3. Exemplos representativos de COX / SDH dupla rotulagem. Seções do cérebro de tipo selvagem e camundongos mutator prematuramente envelhecimento mtDNA foram seqüencialmente marcados para as atividades COX e SDH. (Bar da escala: 200. Mm) a atividade da COX Normal (indicada pela cor marrom escuro) foi mostrado no hipocampo de camundongos selvagens (esquerda). Deficiências COX (indicado pela cor azul) foram revelados no hipocampo de ratos mtDNA mutator (centro e direita). Houve uma redução ainda maior na atividade da COX por 46 semanas de idade em ratos mutator mtDNA, sugerindo exacerbação da disfunção generalizada da cadeia respiratória. O mito observadodisfunção mitocondrial em ratos mtDNA mutator 12 é causada por altos níveis de mutações pontuais mtDNA, bem como aumento dos níveis de deleções linear 5. <img alt="Figura 4" src="/files/ftp_upload/3266/3266fig4.jpg" /> Figura 4. Exemplos de inadequado COX / SDH dupla rotulagem. Seções do cérebro de camundongos selvagens foram seqüencialmente marcados para as atividades COX e SDH. (Bar da escala: 200. Mm) tempos de incubação inadequada (10 e 25 minutos) para a demonstração da atividade da COX resultou em uma redução de deposição do produto da reação DAB marrom, em comparação com o tempo de incubação de 40 minutos (esquerda e centro). Os tempos de incubação reduzidos permitiu a formação do produto final formazan azul durante a incubação SDH, enganosamente, sugerindo a presença de células com deficiências COX. Cobertura das lâminas durante a incubação COX também resultou na formação imprecisas e deposição do reagir DABíon produto (direita). <img alt="Figura 5" src="/files/ftp_upload/3266/3266fig5.jpg" /> Figura 5. I INDIVIDUAIS COX e SDH rotulagem e controle de especificidade. Seções do cérebro de camundongos selvagens foram separadamente rotulado para COX e SDH atividades, indicado pela cor castanho escuro ea cor azul, respectivamente (esquerda e centro). Embora as atividades de COX e SDH podem ser individualmente rotulados, os rótulos seqüencial mostrou-se vantajosa em localizar células com disfunção mitocondrial. Um exemplo de um controle de especificidade para COX e SDH atividades no cérebro de um rato do tipo selvagem mostrou falta de rotulagem (direita). (Bar da escala: 200. Mm)

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O método de COX / SDH combinado histoquímica permite a visualização de células com disfunção mitocondrial. Esta técnica, com estudos iniciais datam de 1968, permanece popular, com muitos considerando-o &quot;padrão ouro&quot; para a identificação de doenças mitocondriais em pacientes 14,19,26,27. Agora, é freqüentemente usada para investigar o envelhecimento mutação mtDNA-driven e envelhecimento distúrbios relacionados 12,13,18,20,21,24. A COX / SDH método duplo-rotulagem é frequ...

<table border="1"><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comments (opcional) </td></tr><tr><td> Gelo seco </td><td> AGA Gas AB </td><td> forma de bloco </td><td></td></tr><tr><td> Isopentano (2 metilbutano) </td><td> Sigma-Aldrich </td><td> 277258 CAS: 78-78-4 </td><td></td></tr><tr><td> Cyrostat solução incorporação </td><td> Sakura Finetek </td><td> Tissue Tek 4583 </td><td></td></tr><tr><td> Criostato </td><td> Microm </td><td> Microm Modelo HM 500M </td><td></td></tr><tr><td> Slides </td><td> Thermo Scientific </td><td> Super geada Mais Menzel Glaser J1800AMWZ </td><td></td></tr><tr><td> Lamínulas de vidro Vidro borosilicato </td><td> VWR International </td><td> 16004-098 </td><td> 24 x 50 mm </td></tr><tr><td> Papel de filtro </td><td> Munktell Filtro AB </td><td> Qualidade: 1350 ArtigoNúmero: 242 001 </td><td> 430 x 430 mm </td></tr><tr><td> Tetrahidrocloreto 3,3 &#39;diaminobenzidina (DAB) </td><td> Sigma-Aldrich </td><td> Sigma Sistema de substrato líquido, D7304 </td><td></td></tr><tr><td> Citocromo c (Tipo III, do coração de eqüinos) </td><td> Sigma-Aldrich </td><td> C2506 CAS: 9007-43-6 </td><td></td></tr><tr><td> Bovina catalase (de fígado) </td><td> Sigma-Aldrich </td><td> C9322 CAS: 9001-05-2 </td><td></td></tr><tr><td> Nitroblue tetrazólio (NBT) </td><td> Sigma-Aldrich </td><td> N6876 CAS: 298-83-9 </td><td></td></tr><tr><td> Succinato de sódio </td><td> Sigma-Aldrich </td><td> S2378 CAS: 6106-21-4 </td><td></td></tr><tr><td> Metasulfato fenazina (PMS) </td><td> Sigma-Aldrich </td><td> P9625 CAS: 299-11-6 </td><td> PMS é sensível à luz. Escudo da luz. </td></tr><tr><td> Azida de sódio </td><td> Sigma-Aldrich </td><td> S8032 CAS: 26628-22-8 </td><td></td></tr><tr&gt; <td> Xileno </td><td> VWR International </td><td> EM-XX0060-4 </td><td></td></tr><tr><td> Entellan </td><td> VWR International </td><td> 100503-870 </td><td></td></tr><tr><td> Malonato (Ácido malónico) </td><td> Sigma-Aldrich </td><td> M1296 CAS: 141-82-2 </td><td></td></tr></table>

visualization of mitochondrial respiratory function using cytochrome c oxidase / succinate dehydrogenase (cox/sdh) double-labeling histochemistry

DNA mitocondrial (mtDNA) defeitos são uma importante causa de doença e pode ser a base de envelhecimento e relacionadas ao envelhecimento 1,2 alterações. A teoria mitocondrial do envelhecimento sugere um papel para mutações do mtDNA, que pode alterar a homeostase bioenergética e função celular, no processo de envelhecimento 3. A riqueza de evidências foram compiladas em apoio a este 1,4 teoria, a exemplo do mtDNA modificador do mouse 5, mas o papel preciso de mtDNA danos do envelhecimento não é totalmente compreendido 6,7. Observando a atividade das enzimas respiratórias é uma abordagem simples para investigar a disfunção mitocondrial. Complexo IV ou citocromo c oxidase (COX), é essencial para a função mitocondrial. As subunidades catalítica da COX são codificadas por mtDNA e são essenciais para a montagem do complexo (Figura 1). Assim, a síntese adequada e função são em grande parte com base na integridade mtDNA 2.Apesar de outros complexos respiratórios poderiam ser investigados, Complexos IV e II são as mais passíveis de exame histoquímico 8,9. Complexo II ou succinato desidrogenase (SDH), é inteiramente codificada pelo DNA nuclear (Figura 1), e sua atividade normalmente não é afetado por deficiência mtDNA, apesar de um aumento pode indicar biogênese mitocondrial 10-12. O mtDNA prejudicada observado em doenças mitocondriais, envelhecimento e doenças relacionadas à idade, muitas vezes leva à presença de células com baixa ou ausente a atividade da COX 2,12-14. Embora as atividades de COX e SDH podem ser investigados individualmente, o método duplo-rotulagem seqüencial 15,16 provou ser vantajoso para localizar células com disfunção mitocondrial 12,17-21. Muitas das constituições ótima do ensaio foram determinadas, como a concentração do substrato, receptores de elétrons / doadores, intermediário portadores de elétrons, a influência do pH e de reação time 9,22,23. 3,3 &#39;-diaminobenzidina (DAB) é um doador de elétrons eficaz e confiável 22. Em células com funcionamento COX, o indamine marrom produto polímero irá localizar em cristas mitocondriais e saturar as células 22. Essas células com COX disfuncionais, portanto, não ser saturado pelo produto DAB, permitindo a visualização da atividade SDH pela redução da nitroblue tetrazólio (NBT), um receptor de elétrons, a um produto azul final formazan 9,24. Citocromo c e substratos succinato de sódio são adicionados para normalizar os níveis endógenos entre controle e doentes / mutante tecidos 9. Catalase é adicionado como uma precaução para evitar possíveis reações contaminação da atividade da peroxidase 9,22. Metasulfato fenazina (PMS), uma transportadora de elétrons intermediário, é utilizado em conjunto com azida de sódio, um inibidor da cadeia respiratória, para aumentar a formação dos produtos da reacção final de 9,25. Apesar desta informarção, alguns detalhes críticos que afetam o resultado deste ensaio seemly simples, além de controles especificidade e avanços na técnica, ainda não foram apresentados.

Watch this Scientific Journal Video about Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry at JoVE.com

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry

O citocromo c oxidase desidrogenase / sódio (COX / SDH) método duplo-rotulagem permite a visualização direta de deficiências enzimáticas mitocondriais respiratória em seções de tecido fresco congelado. Esta é uma técnica simples histoquímica e é útil na investigação de doenças mitocondriais, envelhecimento, envelhecimento e distúrbios relacionados.

Visualização da função respiratória mitocondrial usando Citocromo C Desidrogenase Oxidase / Succinato (COX / SDH) Histoquímica rotulagem dupla

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of ...

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Research

JoVE Journal

Biology

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry

36.5K visualizações.  Karolinska Institutet. O citocromo c oxidase desidrogenase / sódio (COX / SDH) método duplo-rotulagem permite a visualização direta de deficiências enzimáticas mitocondriais respiratória em seções de tecido fresco congelado. Esta é uma técnica simples histoquímica e é útil na investigação de doenças mitocondriais, envelhecimento, envelhecimento e distúrbios relacionados.

Vídeo: Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

Geração de transgênicos Estável C. elegans Usando microinjeção

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

Biologia

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

Ativação da apoptose por microinjeção citoplasmática de citocromo C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

The isolated microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of factors that control vessel diameter, and thus, perfusion resistance1-5. This is a classic experimental preparation that was, in large measure, initially described by Uchida et al.15 several decades ago. This initial description provided the basis for the techniques that was extensively modified and enhanced, primarily in the laboratory of Dr. Brian Duling at the University of Virginia6-8, and we present a current approach in the following pages. This preparation will specifically refer to the gracilis arteriole in a rat as the microvessel of choice, but the basic preparation can readily be applied to vessels isolated from nearly any other tissue or organ across species9-13. Mechanical (i.e., dimensional) changes in the isolated microvessels can easily be evaluated in response to a broad array of physiological (e.g., hypoxia, intravascular pressure, or shear) or pharmacological challenges, and can provide insight into mechanistic elements comprising integrated responses in an intact, although ex vivo, tissue. The significance of this method is that it allows for facile manipulation of the influences on the integrated regulation of microvessel diameter, while also allowing for the control of many of the contributions from other sources, including intravascular pressure (myogenic), autonomic innervation, hemodynamic (e.g., shear stress), endothelial dependent or independent stimuli, hormonal, and parenchymal influences, to provide a partial list. Under appropriate experimental conditions and with appropriate goals, this can serve as an advantage over in vivo or in situ tissue/organ preparations, which do not readily allow for the facile control of broader systemic variables. 
The major limitation of this preparation is essentially the consequence of its strengths. By definition, the behavior of these vessels is being studied under conditions where many of the most significant contributors to the regulation of vascular resistance have been removed, including neural, humoral, metabolic, etc. As such, the investigator is cautioned to avoid over-interpretation and extrapolation of the data that are collected utilizing this preparation. The other significant area of concern with regard to this preparation is that it can be very easy to damage cellular components such as the endothelial lining or the vascular smooth muscle, such that variable source of error can be introduced. It is strongly recommended that the individual investigator utilize appropriate measurements to ensure the quality of the preparation, both at the initiation of the experiment and periodically throughout the course of a protocol.

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

An ex vivo preparation is described for isolation of the largest gracilis muscle resistance arterioles for interrogation of both vascular responses to vasoactive stimuli and the assessment of basic structural properties via passive wall mechanics.

O Ex vivo Preparação microvasos isolada esquelético de Investigação da reatividade vascular

The isolated  microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of  factors that control vessel ...

Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC regulates mitochondrial function and cellular energy status. Numerous reports demonstrated that the activation of PKC-a and PKC-&epsilon; improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-&alpha; and PKC-&epsilon; are involved in nephrotoxicant-induced mitochondrial dysfunction and cell death in kidney cells. Therefore, the goal of this study was to develop an in vitro model of renal cells maintaining active mitochondrial functions in which PKC isozymes could be selectively activated or inhibited to determine their role in regulation of oxidative phosphorylation and cell survival. Primary cultures of renal proximal tubular cells (RPTC) were cultured in improved conditions resulting in mitochondrial respiration and activity of mitochondrial enzymes similar to those in RPTC in vivo. Because traditional transfection techniques (Lipofectamine, electroporation) are inefficient in primary cultures and have adverse effects on mitochondrial function, PKC-&epsilon; mutant cDNAs were delivered to RPTC through adenoviral vectors. This approach results in transfection of over 90% cultured RPTC.
Here, we present methods for assessing the role of PKC-&epsilon; in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-&epsilon; is activated by overexpressing the constitutively active PKC-&epsilon; mutant. PKC-&epsilon; is inhibited by overexpressing the inactive mutant of PKC-&epsilon;. Mitochondrial function is assessed by examining respiration, integrity of the respiratory chain, activities of respiratory complexes and F0F1-ATPase, ATP production rate, and ATP content. Respiration is assessed in digitonin-permeabilized RPTC as state 3 (maximum respiration in the presence of excess substrates and ADP) and uncoupled respirations. Integrity of the respiratory chain is assessed by measuring activities of all four complexes of the respiratory chain in isolated mitochondria. Capacity of oxidative phosphorylation is evaluated by measuring the mitochondrial membrane potential, ATP production rate, and activity of F0F1-ATPase. Energy status of RPTC is assessed by determining the intracellular ATP content. Mitochondrial morphology in live cells is visualized using MitoTracker Red 580, a fluorescent dye that specifically accumulates in mitochondria, and live monolayers are examined under a fluorescent microscope. RPTC viability is assessed using annexin V/propidium iodide staining followed by flow cytometry to determine apoptosis and oncosis.
These methods allow for a selective activation/inhibition of individual PKC isozymes to assess their role in cellular functions in a variety of physiological and pathological conditions that can be reproduced in in vitro.

The effects of activation of protein kinase C (PKC) isozymes on mitochondrial functions associated with respiration and oxidative phosphorylation and on cell viability are described. The approach adapts adenoviral technique to selectively overexpress PKC isozymes in primary cell culture and a variety of assays to determine mitochondrial functions and energy status of the cell.

Avaliação de funções mitocondriais e viabilidade celular em células renais Proteína Kinase C superexpressão Isozimas

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC ...

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.

Visualization of experimental data has become a key element in presenting results to the scientific community. Generation of live time-lapse recording of growing embryos contributes to better presentation and understanding of complex developmental processes. This protocol is a step-by-step guide to cell labeling via photoconversion of kaede protein in zebrafish.

Visualização de Desenvolvimento Craniofacial nas SOX10: Kaede Transgênicos Zebrafish Linha Usando Time-lapse microscopia confocal

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well when a small number of genes are targeted for knockdown, large scale feeding screens show variable knockdown efficiencies, which limits their utility. We have deconstructed previously published RNAi knockdown protocols and found that the primary source of the reduced knockdown can be attributed to the loss of dsRNA-encoding plasmids from the bacteria fed to the animals. Based on these observations, we have developed a dsRNA feeding protocol that greatly reduces or eliminates plasmid loss to achieve efficient, high throughput knockdown. We demonstrate that this protocol will produce robust, reproducible knock down of C. elegans genes in multiple tissue types, including neurons, and will permit efficient knockdown in large scale screens. This protocol uses a commercially available dsRNA feeding library and describes all steps needed to duplicate the library and perform dsRNA screens. The protocol does not require the use of any sophisticated equipment, and can therefore be performed by any C. elegans lab.

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

While dsRNA feeding in C. elegans is a powerful tool to assess gene function, current protocols for large scale feeding screens result in variable knockdown efficiencies. We describe an improved protocol for performing large scale RNAi feeding screens that results in highly efficacious and reproducible knockdown of gene expression.

Em larga escala Knockdown Gene em<em&gt; C. elegans</em&gt; Usando bibliotecas de alimentação dsRNA para gerar fenótipos robustos perda de função

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well ...

Measuring Respiratory Function in Mice Using Unrestrained Whole-body Plethysmography

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative assessment of lung function in rodent models is an important tool in the development of future therapies. Commonly used techniques for assessing respiratory function including invasive plethysmography and forced oscillation. While these techniques provide valuable information, data collection can be fraught with artefacts and experimental variability due to the need for anesthesia and/or invasive instrumentation of the animal. In contrast, unrestrained whole-body plethysmography (UWBP) offers a precise, non-invasive, quantitative way by which to analyze respiratory parameters. This technique avoids the use of anesthesia and restraints, which is common to traditional plethysmography techniques. This video will demonstrate the UWBP procedure including the equipment set up, calibration and lung function recording. It will explain how to analyze the collected data, as well as identify experimental outliers and artefacts that results from animal movement. The respiratory parameters obtained using this technique include tidal volume, minute volume, inspiratory duty cycle, inspiratory flow rate and the ratio of inspiration time to expiration time. UWBP does not rely on specialized skills and is inexpensive to perform. A key feature of UWBP, and most appealing to potential users, is the ability to perform repeated measures of lung function on the same animal.

The assessment of respiratory physiology has traditionally relied upon techniques, which require restraint or sedation of the animal. Unrestrained whole-body plethysmography, however, provides precise, non-invasive, quantitative analysis of respiratory physiology in animal models. In addition, the technique allows repeated respiratory assessment of mice allowing for longitudinal studies.

Medir a função respiratória em ratos usando Unrestrained pletismografia de corpo inteiro

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative ...

High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in biological samples (intact and permeabilized cells, tissues or isolated mitochondria). The high-resolution oxygraph device is equipped with two chambers and uses polarographic oxygen sensors to measure oxygen concentration and calculate oxygen consumption within each chamber. Oxygen consumption rates are calculated using software and expressed as picomoles per second per number of cells. Each high-resolution oxygraph chamber contains a stopper with injection ports, which makes it ideal for substrate-uncoupler-inhibitor titrations or detergent titration protocols for determining effective and optimum concentrations for plasma membrane permeabilization. The technique can be applied to measure respiration in a wide range of cell types and also provides information on mitochondrial quality and integrity, and maximal mitochondrial respiratory electron transport system capacity.

High-resolution respirometry is used to determine mitochondrial oxygen consumption. This is a straightforward technique to determine mitochondrial respiratory chain complexes&#39; (I-IV) respiratory rates, maximal mitochondrial electron transport system capacity, and mitochondrial outer membrane integrity.

De alta resolução Respirometria para avaliar a função mitocondrial em permeabilizadas e células intactas

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in ...

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Fluorescence microscopy is an indispensable tool for the direct assessment of mitochondrial structure and function in live cells and for studying the mitochondrial structure-function relationship, which is primarily modulated by the molecules governing fission and fusion events between mitochondria. This paper describes and demonstrates specific methods for studying mitochondrial structure and function in live as well as in fixed tissue in the model organism Drosophila melanogaster. The tissue of choice here is the Drosophila ovary, which can be isolated and made amenable for ex vivo live confocal microscopy. Furthermore, the paper describes how to genetically manipulate the mitochondrial fission protein, Drp1, in Drosophila ovaries to study the involvement of Drp1-driven mitochondrial fission in modulating the mitochondrial structure-function relationship. The broad use of such methods is demonstrated in already-published as well as in novel data. The described methods can be further extended towards understanding the direct impact of nutrients and/or growth factors on the mitochondrial properties ex vivo. Given that mitochondrial dysregulation underlies the etiology of various diseases, the described innovative methods developed in a genetically tractable model organism, Drosophila, are anticipated to contribute significantly to the understanding of the mechanistic details of the mitochondrial structure-function relationship and to the development of mitochondria-directed therapeutic strategies.

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Specific methods for studying mitochondrial structure and function in live and fixed Drosophila ovaries are described and demonstrated in this paper.

Estudar mitocondrial estrutura e função em<em&gt; Drosophila</em&gt; ovários

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.