1. Coleta de tecido: <ol><li> Use ratos entre 7 e 12 semanas de idade. Normalmente, pelo menos dois animais são utilizados: um é para configurar o FACS cor única controles necessários para compensação e de fluorescência-menos e uma amostras controle isotipo. A outra é a amostra real para a classificação. </li><li> Eutanásia os ratos por inalação de CO 2 ou de acordo com um protocolo de ética de escolha. Camundongos lugar em uma toalha de papel, virada para cima, e pulverize com etanol 70% completamente para evitar a contaminação com a pele do rato. </li><li> Levante a pele da região abdominal com uma pinça, faça um pequeno corte longitudinal com tesouras afiadas. </li><li> Descasque as duas abas de pele em sentidos opostos (para cima e para baixo) para expor completamente os músculos dos membros posteriores por baixo. </li><li> O tecido muscular especiais de consumo, inserindo e ampliando a tesoura ao longo de ambos os lados da tíbia. </li><li> Repita o mesmo processo para o fêmur para extrair o tecido muscular. </li><li> Coloque todos os tecidos musculares excisadas em 60 milímetros petri pratos, usando um prato para o tecido de cada rato. </li><li> Repita o procedimento acima para todos os ratos. </li></ol> 2. Dissociar tecido em células isoladas por digestão enzimática: Ao longo desta secção do protocolo, o uso de reagentes estéreis e ferramentas de trabalho e em condições estéreis. <ol start="9"><li> Adicionar 2 mL de colagenase II (Sigma, cat # 6885, 500U / mL) e 20 mL de CaCl 2 250mm de ações para cada placa de Petri. </li><li> Rasgar o tecido muscular em pedaços pequenos (cerca de 1 mm 3) com duas pinças (uma dentada, um com 2 dentes, a partir de Ferramentas Ciência Fine, cat # 11051-10, 11154-10), prestando atenção ao remover e descartar tanto contaminados não-tecido muscular possível. </li><li> Cobrir os pratos de Petri e incube-os a 37 graus Celsius por 30 minutos. </li><li> Recuperar os pratos de Petri e usar um êmbolo da seringa 3 mL para triture bem os pedaços de tecido. Use um êmbolo para cada amostra para evitar contaminações. </li><li> Adicione um pouco de (~ 5 mL) estéril fria PBS a cada Placa de Petri e transferência do lodo de tecido em um tubo Falcon 50 ml (lavar o prato Petri-se necessário para recuperar todo o tecido) </li><li> Agite vigorosamente o tubo Falcon, encha o tubo com PBS, centrifugue @ 800rpm x 5 minutos (Eppendorf modelo de bancada: 5810R) e aspirar o sobrenadante (repete 3 vezes) </li><li> Adicione a mistura de 1 ml de colagenase D (Roche, Cat # 11088882001, 1.5u / mL) / Dispase II (Roche, Cat #: 04942078001, 2.4u / mL) e 10μL de CaCl 2 (250mm) para cada tubo, incubar a 37 graus Celsius com a rotação durante uma hora. </li><li> Adicionar 5-10 mL de frio estéril PBS, vortex, e pipetar cuidadosamente para homogeneizar o tecido. </li><li> Encha os tubos com PBS e misture bem. </li><li> Transferir o líquido para um filtro de células 40μm colocado em cima de um tubo de Falcon 50 mL para filtrar os pedaços restantes grandes de tecido. Divida cada amostra uniformemente em três tubos de 50mL Falcon. Encha cada um dos tubos com PBS, em seguida, centrifugar @ 1600rpm por 5 minutos a 8 graus Celsius. </li><li> Desprezar o sobrenadante e coletar células para a coloração. </li></ol> 3. Coloração de anticorpos e classificação: Ao longo desta secção do protocolo, use FACS estéril e buffers de coleta e de trabalho em condições estéreis. Anticorpos comerciais são geralmente considerados estéreis se for manuseado correctamente. <ol start="20"><li> Para única cor e amostras de controle isotipo, ressuspender a amostra em tampão FACS 1mL e dividir as células suspensas em nove tubos de 1,5 ml pré-rotulados Eppendorf (100 mL cada tubo). <ol><li> Configurar coloração única cor mistura de acordo com a tabela a seguir: ATENÇÃO: A concentração final de anticorpos isotipo controle na mancha deve coincidir com o anticorpo primário são o controle de. Por exemplo, se anti-Sca-1 é usado para uma μg/10 6 células, o seu isotipo correspondente deverá ser usado na mesma concentração. <table border="1"><tbody><tr><td> Tubo # </td><td> Nome do tubo </td><td> Companhia </td><td> Cat # </td><td> clone </td><td> isotipo </td><td> diluição </td></tr><tr><td> 1 </td><td> Não mancha </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> 2 </td><td> Hoechst33342 </td><td> sigma </td><td> B2261 </td><td></td><td></td><td> 2-5ug/ml </td></tr><tr><td> 3 </td><td> PI </td><td> sigma </td><td> P4864 </td><td></td><td></td><td> 1ug/ml </td></tr><tr><td rowspan="2"> 4 </td><td rowspan="2"> CD31-FITC CD45-FITC </td><td> eBioscience </td><td> 11-0311-85 </td><td> 390 </td><td> Rat IgG2a </td><td> 500 </td></tr><tr><td> Casa feita </td><td></td><td> I3 / 2 </td><td> Rat IgG2b </td><td> 500 </td></tr><tr><td> 5 </td><td> SCA1-PECY7 </td><td> eBioscience </td><td> 25-5981-82 </td><td> D7 </td><td> Rat IgG2a </td><td> 5000 </td></tr><tr><td> 6 </td><td> α-7 APC </td><td> Casa feita </td><td></td><td> R2F2 </td><td> Rat IgG2b </td><td> 1000 </td></tr></tbody></table></li><li> Configurar isotipo coloração controle mistura usando a seguinte tabela: <table border="1"><tbody><tr><td> Tubo # </td><td> Nome do tubo </td><td> Hoechst </td><td> PI </td><td> CD31- FITC </td><td> CD45- FITC </td><td> Scal- PE-Cy7 </td><td> A-7- APC </td><td> Rat- IgG2b- APC </td><td> Rat- IgG2a- pecy7 </td><td> Rat- IgG2a- FITC </td><td> Rat- IgG2b- FITC </td></tr><tr><td> 7 </td><td> Iso- CD31 / CD45 </td><td> + </td><td> + </td><td> - </td><td> - </td><td> + </td><td> + </td><td> - </td><td> - </td><td> + </td><td> + </td></tr><tr><td> 8 </td><td> Iso- Scal- pecy7 </td><td> + </td><td> + </td><td> + </td><td> + </td><td> - </td><td> + </td><td> - </td><td> + </td><td> - </td><td> - </td></tr><tr><td> 9 </td><td> Iso- α-7-APC </td><td> + </td><td> + </td><td> + </td><td> + </td><td> + </td><td> - </td><td> + </td><td> - </td><td> - </td><td> - </td></tr></tbody></table></li><li> Pipeta a única cor e mistura de isótopos de controle de marcação (que geralmente ajustar a concentração de anticorpos, para que entre 5 e 10 mL de coloração mix são adicionados a cada amostra) para o tubo Eppendorf apropriado a partir do Passo 20. Misturar bem e incubar no gelo por 1 hora. </li></ol></li><li> Mancha células de rato amostra com 800μL do coquetel de anticorpos (abaixo) para separação de células FACS. Misturar bem e incubar no gelo por 1 hora. Cocktail de anticorpos: Hoechst, PI, CD31-FITC, CD45-FITC, SCA1-PECY7, α-7 APC ATENÇÃO: A proporção ideal de anticorpos para as células deve ser determinada por titulação individualmente cada anticorpo, como diferenças significativas possam existir entre os lotes. A quantidade ideal de anticorpos para ser usado deve ser expresso em μgs de anticorpos por 10 6 células-alvo e mantida constante quando se utiliza o mesmo lote de anticorpos. Claramente, se o número de células alvo não muda significativamente entre experimentos, ou se o volume coloração é escalado com o número de células, uma diluição fixa de cada anticorpo também pode ser usado. </li><li> Lavagem: Adicionar cerca de 1mL de tampão FACS a cada um dos nove tubos de controle, adicionar cerca de 20mL de tampão FACS para o tubo de amostra. </li><li> Centrifugar a tubos Eppendorf @ 3000rpm por 5 minutos (bancada topo centrífuga Eppendorf modelo: 5417C). Centrifugar o tubo de amostra @ 1600rpm por 5 minutos (bancada topo centrífuga Eppendorf modelo: 5810R). </li><li> Aspirar e desprezar o sobrenadante de todos os tubos. </li><li> Ressuspender as células no buffer FACS (1mL para cada um dos controles, 4 ml da amostra de classificação), células Pipetar em &quot;tubos FACS&quot; através do filtro de células cap (BD gato Falcon # 352235, 40μm tamanho dos poros) para remover pedaços restantes que podem afetar o citômetro. </li><li> Configure o Sorter FACS com controles de cor única e controles isotipo. </li><li> Coletar células classificados de acordo com a tabela a seguir em um buffer de coleta de 3,5 ml (95% DMEM, 5% FBS) separadamente. Normalmente, quando a colheita ambos os membros posteriores, um único tipo de rato silvestre poderia render cerca de 150.000 células progenitoras miogênica ou 100.000 células progenitoras adipogénicos, com a viabilidade acima de 95%, a avaliar pela re-análise de células classificadas por FACS, no final do procedimento (Figura 1 ) Definição de progenitores miogênicos e adipogénicos baseado em manchas na superfície <table border="1"><tbody><tr><td> Antibody / mancha </td><td> Progenitoras miogênica (MP) </td><td> Progenitoras adipogénicos (AP) </td></tr><tr><td> Hoechst </td><td> + </td><td> + </td></tr><tr><td> PI </td><td> - </td><td> - </td></tr><tr><td> CD31-FITC </td><td> - </td><td> - </td></tr><tr><td> CD45-FITC </td><td&gt; - </td><td> - </td></tr><tr><td> SCA1-PECY7 </td><td> - </td><td> + </td></tr><tr><td> α-7 APC </td><td> + </td><td> - </td></tr></tbody></table></li></ol> 4. Transplante: <ol start="28"><li> Centrifugar as células classificadas @ 1500rpm por 5 minutos, retire as células sobrenadante e transferir para autoclavado tubos Eppendorf (1,5 mL), lavar as células com 500 mL de PBS estéril (soro grátis!), Então centrífuga @ 3000rpm por 5 minutos. Manutenção de todos os reagentes em condições estéreis e trabalhando em uma capa de cultura de tecidos, ressuspender progenitores miogênicos em PBS, ou progenitor adipogénicos em Matrigel (BD cat # 354234) em aproximadamente 10 6 células / mL. </li><li> Transplante de células progenitoras ou miogênica progenitor adipogénicos a camundongos destinatário. <ol><li> Anestesiar o rato com isoflouran acordo com sua política instituição. </li><li> Raspar o cabelo em torno da região de injeção e, em seguida esterilizar a pele por fricção com álcool a 70% </li><li> Injetar 20μL de células progenitoras ou células progenitoras miogênica adipogénicos (= 20k células) usando um 10/03 seringa de insulina CC (BD cat # 309300). </li></ol></li><li> Depois de um tempo adequado (três semanas funciona bem, mas miofibras expressar doadores marcadores derivados transgênicos são geralmente evidente dentro de uma semana após o transplante), anestesiar os ratos destinatário a partir do passo 29 por injeção intraperitoneal de 400 mL Avertin (Sigma Cat # T04840-2, 25 mg / mL), então perfundir transcardially com 50mL de PBS (contém 10 mM de EDTA) em primeiro lugar, seguido de 15ml de PFA 4%. Coletar tecido alvo, pós-fix com PFA 4% durante a noite, se necessário, então a transferência para sacarose a 20% durante a noite. Incorporar em outubro congelar, e cryosection. </li></ol> 5. Resultados representativos: MP quando são transplantadas no músculo esquelético do rato, eles prontamente se fundem com fibras musculares pré-existentes. Portanto, qualquer rótulo genético presente nas células do doador serão facilmente detectáveis ​​nas fibras que os recebeu. A Figura 2 mostra um exemplo onde as células transplantadas expressa phostphatase alcalinas humanos, revelou histoquimicamente. AP quando são transplantadas o resultado é altamente dependente do ambiente do local de transplante: quando transplantadas por via subcutânea essas células dão origem aos adipócitos e miofibroblastos (ver figura 2). Em muitos outros sites, incluindo o músculo, não sobreviver, a menos degeneração gordurosa foi induzida 3. <img src="/files/ftp_upload/2476/2476fig1.jpg" alt="Figura 1" /> Figura 1. Classificação estratégia para o isolamento de populações de células progenitoras do músculo esquelético (A) estratégia de classificação:. Células viáveis ​​foram identificados com base na dispersão para a frente e dispersão lateral. Coloração Hoechst foi usada para excluir restos anuclear e propidium iodeto de coloração (PI) para excluir as células mortas. Hematopoiéticas (CD45) e endoteliais (CD31), as células foram excluídos os portões de classificação. O α7 + + Hoechst PI - CD45 - CD31 - scal - subconjunto contém todos os progenitores miogênicos (MP). O scal + + Hoechst PI - CD45 - CD31 - α7 - População contém progenitores adipogénicos (AP). (B) de fluorescência-menos-um controles (FMO) isotipo confirmar a especificidade da mancha. (C) Pureza cheques de subconjuntos MP e AP após a triagem. <img src="/files/ftp_upload/2476/2476fig2.jpg" alt="Figura 2" /> Figura 2. Resultados representativos seguintes MP e transplante AP. AP foram transplantados subcutaneamente (painel superior) e MP por via intramuscular (painel inferior). Em ambos os casos, as células do doador se originou de um camundongo transgênico expressando fosfatase alcalina humana, identificados pela coloração marrom.

<ol>
	<li>Buckingham, M., Montarras, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+stem+cells.">Skeletal muscle stem cells.</a> Curr Opin Genet Dev. 18, 330-336 (2008).</li><li>Relaix, F., Marcelle, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+stem+cells.">Muscle stem cells.</a> Curr Opin Cell Biol. 21, 748-753 (2009).</li><li>Joe, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+injury+activates+resident+fibro/adipogenic+progenitors+that+facilitate+myogenesis.">Muscle injury activates resident fibro/adipogenic progenitors that facilitate myogenesis.</a> Nat Cell Biol. 12, 153-163 (2010).</li><li>Natarajan, A., Lemos, D. R., Rossi, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibro/adipogenic+progenitors:+A+double-edged+sword+in+skeletal+muscle+regeneration.">Fibro/adipogenic progenitors: A double-edged sword in skeletal muscle regeneration.</a> Cell Cycle. 9, (2010).</li><li>Sherwood, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+adult+mouse+myogenic+progenitors:+functional+heterogeneity+of+cells+within+and+engrafting+skeletal+muscle.">Isolation of adult mouse myogenic progenitors: functional heterogeneity of cells within and engrafting skeletal muscle.</a> Cell. 119, 543-554 (2004).</li><li>Sacco, A., Doyonnas, R., Kraft, P., Vitorovic, S., Blau, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-renewal+and+expansion+of+single+transplanted+muscle+stem+cells.">Self-renewal and expansion of single transplanted muscle stem cells.</a> Nature. , (2008).</li><li>Montarras, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+isolation+of+satellite+cells+for+skeletal+muscle+regeneration.">Direct isolation of satellite cells for skeletal muscle regeneration.</a> Science. 309, 2064-2067 (2005).</li></ol>

Não há conflitos de interesse declarados.

O objetivo deste protocolo é conseguir um equilíbrio razoável entre os rendimentos elevados e alta viabilidade das células purificadas. O passo mais crítico para garantir que as células saudáveis ​​são recuperados é, previsivelmente, a dissociação enzimática do tecido de partida. O manuseio do tecido deve ser particularmente delicado, que é difícil de demonstrar ou apreciar ainda em um formato audiovisual. Outro fator importante é a duração do procedimento. O tempo que leva para ir do doador para o animal receptor, menor a viabilidade e, portanto, a eficiência de enxerto. Caso haja qualquer dúvida sobre a viabilidade de que não é imediatamente respondeu, olhando para a frequência de PI evento positivo nas amostras de células purificadas depois da classificação, sugerimos que um ensaio por diluição limitante para medir clonogenicity é realizada 3. Espécie típica do músculo danificado rotineiramente produzir cerca de 1 em 15-20 células capazes de iniciar colônias miogênica ou fibro / adipogénicos in vitro. Embora essencialmente 100% das colônias iniciadas a partir de MPs contêm miotubos diferenciados, apenas cerca de um terço das colônias obtidas FAPs conter adipócitos, além de miofibroblastos actina músculo liso positivos.

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários </th></tr><tr><td> Colagenase II </td><td> Sigma-Aldrich </td><td> C-6885, </td><td> 500 u / ml </td></tr><tr><td> Colagenase D </td><td> Roche </td><td> 11088882001 </td><td> 1,5 u / ml </td></tr><tr><td> Deslocar II </td><td> Roche </td><td> 04942078001 </td><td> 2,4 u / ml </td></tr><tr><td> coador celular </td><td> BD </td><td> 352340 </td><td> 40 mM </td></tr><tr><td> Tubo com filtro de células </td><td> BD </td><td> 352235 </td><td> 40 mM </td></tr><tr><td> Hoechst33342 </td><td> Sigma-Aldrich </td><td> B2261 </td><td> 2-5 ug / ml </td></tr><tr><td> CD31-FITC </td><td> Ebiosciences </td><td> 11-0311-85 </td><td> Clone 390 </td></tr><tr><td> CD45-FITC </td><td> Home made </td><td></td><td> Clone I3 / 2 </td></tr><tr><td> SCA1-PECY7 </td><td> Ebiosciences </td><td> 25-5981-82 </td><td> Clone D7 </td></tr><tr><td> α-7 integrina APC </td><td> Home made </td><td> Informe-se com Rossi laboratório </td><td> R2F2 </td></tr></tbody></table>

purification of progenitors from skeletal muscle

Músculo esquelético contém populações progenitoras múltiplas de diferentes origens embrionárias e potencial de desenvolvimento. Progenitores miogênicos, normalmente residente em uma &quot;posição de células satélite&quot; entre a membrana plasmática fibromuscular e da membrana basal laminina-rico que embainha-lo, são auto-renovação de células que é o único comprometido com a linhagem miogênica 1,2. Descrevemos recentemente uma segunda classe de células progenitoras navio associados que podem gerar miofibroblastos e adipócitos brancos, que responde a danos causados ​​por entrar de forma eficiente a proliferação e fornece suporte tróficos para as células miogênicas durante a regeneração do tecido 3,4. Um dos ensaios mais confiável para determinar o potencial de desenvolvimento e regeneração de uma população determinada célula depende de seu isolamento e transplante 5-7. Para este fim temos protocolos otimizados para a sua purificação por citometria de fluxo a partir do músculo enzimaticamente dissociada, que vamos delinear neste artigo. As populações obtidas com este método irá conter células miogênicas colônia ou fibro / adipogénicos formando: não outros tipos de células são capazes de expansão in vitro ou sobrevivendo na entrega vivo. No entanto, quando essas populações são usadas imediatamente após a classificação para a análise molecular (por exemplo qRT-PCR) é preciso ter em mente que os subconjuntos recém-ordenados podem conter outras células contaminante que não têm a capacidade de formar colônias ou enxertando destinatários.

Watch this Scientific Journal Video about Purification of Progenitors from Skeletal Muscle at JoVE.com

Purification of Progenitors from Skeletal Muscle

Método para a dissociação enzimática, rotulagem superfície e purificação por citometria de fluxo de fibro / adipogénicos e progenitores miogênica de músculo esquelético murino.

Purificação de Progenitores do músculo esquelético

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

purification-of-progenitors-from-skeletal-muscle

Research

JoVE Journal

Biology

18.2K visualizações.  University of British Columbia. Método para a dissociação enzimática, rotulagem superfície e purificação por citometria de fluxo de fibro / adipogénicos e progenitores miogênica de músculo esquelético murino.

Vídeo: Purification of Progenitors from Skeletal Muscle

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Adultas e embrionárias Cultura Microexplant Músculo Esquelético e isolamento de células-tronco musculares

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Biologia

Mitochondrial Isolation from Skeletal Muscle

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and regulate a number of signaling cascades2,3. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart4,5. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles.
Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles 6. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37&deg;C7. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 &mu;g) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 &mu;M) is added to start state 3. Oligomycin (1 &mu;M), an ATPase synthase blocker, is used to estimate state 4. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 &mu;M) is added to measurestate 5, or uncoupled respiration 6. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR &ge;4 is considered as evidence of a viable mitochondria preparation.
In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour.

Isolamento mitocondrial do músculo esquelético

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and ...

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 ...

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see 1). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel2 or sapje3) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes4. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy5. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally1. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo6, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells7.
Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.
Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail8, 9. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish10, 11.

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

Laser-infligido Lesão de Zebrafish muscular esquelético embrionário

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Isolamento e Cultura de miofibras individuais e suas células satélites do músculo esquelético

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

Isolamento de Blood-derivado do navio Multipotentes Precursores do músculo-esquelético humano

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the isolation of these vesicles, including ultracentrifugation, ultrafiltration, and size exclusion chromatography. However, not all are suitable for large scale exosome purification and characterization. Outlined here is a protocol for establishing cultures of primary fibroblasts isolated from adult mouse skeletal muscles, followed by purification and characterization of exosomes from the culture media of these cells. The method is based on the use of sequential centrifugation steps followed by sucrose density gradients. Purity of the exosomal preparations is then validated by western blot analyses using a battery of canonical markers (i.e., Alix, CD9, and CD81). The protocol describes how to isolate and concentrate bioactive exosomes for electron microscopy, mass spectrometry, and uptake experiments for functional studies. It can easily be scaled up or down and adapted for exosome isolation from different cell types, tissues, and biological fluids.

This protocol illustrates the 1) the isolation and culture of primary fibroblasts from the adult mouse gastrocnemius muscle as well as 2) purification and characterization of exosomes using a differential ultracentrifugation method combined with sucrose density gradients followed by western blot analyses.

Isolamento e Caracterização de Exossomos de Fibroblastos de Músculo Esquelético

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the ...

Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors (FAPs) and Myogenic Progenitors (MPs) in Skeletal Muscle in the Rat

Fibro-adipogenic Progenitors (FAPs) are resident interstitial cells in skeletal muscle that, together with myogenic progenitors (MPs), play a key role in muscle homeostasis, injury, and repair. Current protocols for FAPs identification and isolation use flow cytometry/fluorescence-activated cell sorting (FACS) and studies evaluating their function in vivo to date have been undertaken exclusively in mice. The larger inherent size of the rat allows for a more comprehensive analysis of FAPs in skeletal muscle injury models, especially in severely atrophic muscle or when investigators require substantial tissue mass to conduct multiple downstream assays. The rat additionally provides a larger selection of muscle functional assays that do not require animal sedation or sacrifice, thus minimizing morbidity and animal use by enabling serial assessments. The flow cytometry/FACS protocols optimized for mice are species specific, notably restricted by the characteristics of commercially available antibodies. They have not been optimized for separating FAPs from rat or highly fibrotic muscle. A flow cytometry/FACS protocol for the identification and isolation of FAPs and MPs from both healthy and denervated rat skeletal muscle was developed, relying on the differential expression of surface markers CD31, CD45, Sca-1, and VCAM-1. As rat-specific, flow cytometry-validated primary antibodies are severely limited, in-house conjugation of the antibody targeting Sca-1 was performed. Using this protocol, successful Sca-1 conjugation was confirmed, and flow cytometric identification of FAPs and MPs was validated by cell culture and immunostaining of FACS-isolated FAPs and MPs. Finally, we report a novel FAPs time-course in a prolonged (14 week) rat denervation model. This method provides the investigators the ability to study FAPs in a novel animal model.

Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors FAPs and Myogenic Progenitors MPs in Skeletal Muscle in the Rat

This protocol outlines a method to isolate Fibro-adipogenic progenitors (FAPs) and myogenic progenitors (MPs) from rat skeletal muscle. Utilization of the rat in muscle injury models provides increased tissue availability from atrophic muscle for the analysis and a larger repertoire of validated methods to assess muscle strength and gait in free-moving animals.

Identificação, Isolamento e Caracterização de Progenitores Fibro-Adipogênicos (FAPs) e Progenitores Miogênicos (MPs) em Músculo Esquelético no Rato

Fibro-adipogenic Progenitors (FAPs) are resident interstitial cells in skeletal muscle that, together with myogenic progenitors (MPs), play a key role in ...

Isolation of Mitochondria from Mouse Skeletal Muscle for Respirometric Assays

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the mitochondrial oxidative phosphorylation (OXPHOS) system, which is regulated in response to cellular demands. The lipid molecule Coenzyme Q (CoQ) is essential in this process by transferring electrons to complex III in the electron transport chain (ETC) through constant oxidation/reduction cycles. Mitochondria status and, ultimately, cellular health can be assessed by measuring ETC oxygen consumption using respirometric assays. These studies are typically performed in established or primary cell lines that have been cultured for several days. In both cases, the respiration parameters obtained may have deviated from normal physiological conditions in&#160;any given organ or tissue.
Additionally, the intrinsic characteristics of cultured single fibers isolated from skeletal muscle impede this type of analysis. This paper presents an updated and detailed protocol for the analysis of respiration in freshly isolated mitochondria from mouse skeletal muscle. We also provide solutions to potential problems that could arise at any step of the process. The method presented here could be applied to compare oxygen consumption rates in diverse transgenic mouse models and study the mitochondrial response to drug treatments or other factors such as aging or sex. This is a feasible method to respond to crucial questions about mitochondrial bioenergetics metabolism and regulation.

Here, we describe a detailed method for mitochondria isolation from mouse skeletal muscle and the subsequent analysis of respiration by Oxygen Consumption Rate (OCR) using microplate-based respirometric assays. This pipeline can be applied to study the effects of multiple environmental or genetic interventions on mitochondrial metabolism.

Isolamento das Mitocôndrias do Músculo Esquelético do Rato para Ensaios Respirométricos

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the ...

Electroporation of Plasmid DNA into Mouse Skeletal Muscle

Transient gene expression modulation in murine skeletal muscle by plasmid electroporation is a useful tool for assessing normal and pathological physiology. Overexpression or knockdown of target genes enables investigators to manipulate individual molecular events and, thus, better understand the mechanisms that impact muscle mass, muscle metabolism, and contractility. In addition, electroporation of DNA plasmids that encode fluorescent tags allows investigators to measure changes in subcellular localization of proteins in skeletal muscle in vivo. A key functional assessment of skeletal muscle includes the measurement of muscle contractility. In this protocol, we demonstrate that whole muscle contractility studies are still possible after plasmid DNA injection, electroporation, and gene expression modulation. The goal of this instructional procedure is to demonstrate the step-by-step method of DNA plasmid electroporation into mouse skeletal muscle to facilitate uptake and expression in the myofibers of the tibialis anterior and extensor digitorum longus muscles, as well as to demonstrate that skeletal muscle contractility is not compromised by injection and electroporation.

Electroporation of plasmid DNA into skeletal muscle is a viable method to modulate gene expression without compromising muscle contractility in mice.