Alessandra d'Azzo é titular da Cadeira Joalheira para Crianças (JFC) em Genética e Terapia Gênica. Este trabalho foi apoiado em parte por doações do NIH R01GM104981, RO1DK095169 e CA021764, a Fundação Assis de Memphis e as Instituições de Caridade Associadas Sírias Libanesas Americanas.

Todos os procedimentos em camundongos foram realizados de acordo com protocolos animais aprovados pelo St. Jude Children's Research Hospital, Comitê Institucional de Cuidados e Uso de Animais e diretrizes do National Institutes of Health.
1. Preparação de soluções e meios
<ol><li>Preparar a solução de digestão misturando 15,4 mL de PBS com 2,5 mL de 20 mg/mL de colagenase P (concentração final de 5 mg/mL), 2 mL de dispase II de 11 U/mL (concentração final de 1,2 U/mL) e 100 μL de CaCl2 de 1,0 M (concentração final de 5 mM).</li>
	<li>Preparar 500 mL de meio de fibroblastos primários (DMEM completo) misturando 440 mL de DMEM com 50 mL de FBS (10%), 5,0 mL de pen/strep (100 U/mL e 100 μg/mL, respectivamente) e 5,0 mL de suplemento de glutamina (2 mM). Filtrar-esterilizar o meio com um filtro a vácuo de tamanho de poro de 0,22 μm.</li>
	<li>Preparar soro livre de exossomos por ultracentrifugação noturna de FBS em tubos de polipropileno de 38,5 mL a 100.000 x g a 4 °C e transferir o sobrenadante para um novo tubo.</li>
	<li>Preparar 500 mL de meio livre de exossomos misturando 440 mL de DMEM com 50 mL de soro livre de exossomos (10%), 5 mL de pen/strep (100 U/mL e 100 μg/mL) e 5 mL de suplemento de glutamina (2 mM). Filtrar-esterilizar o meio com um filtro a vácuo de tamanho de poro de 0,22 μm.</li>
	<li>Preparar 0,5 M Tris-HCl (pH 7,4) dissolvendo 6,057 g de Tris base em 90 mL de dH 2 O e ajustando opH para 7,4 com HCl. Ajustar o volume para 100 mL com dH2O.</li>
	<li>Preparar 10 mM de solução de Tris-HCl (pH 7,4)/1 mM Mg(Ac)2 (solução de trabalho de sacarose) misturando 97,9 mL de dH 2 O com 2 mL deTris-HCl 0,5 M (pH = 7,4) e 100 μL de 1 M Mg(Ac)2. Adicione inibidores de protease à solução imediatamente antes de usar e mantenha a solução no gelo.</li>
	<li>Preparar soluções de gradiente de densidade de sacarose no gelo de acordo com a Tabela 1. Essas soluções são suficientes para preparar dois tubos de gradiente de sacarose.</li>
	<li>Preparar 100% de TCA adicionando 40 mL de dH2O a 100 g de pó de TCA e misturar até dissolver completamente. Ajustar o volume final com dH2O para 100 mL.</li>
	<li>Preparar etanol a 80% misturando 20 mL de dH2O com 80 mL de etanol 100%.</li>
	<li>Prepare o buffer de corrida western blot misturando 1,8 L de dH2O com 200 mL de buffer de corrida de 10x.</li>
	<li>Preparar o tampão de transferência western blot misturando 1,4 L de dH2O com 200 mL de tampão de transferência 10x e 400 mL de metanol. Pré-arrefecer o tampão de transferência a 4 °C.</li>
	<li>Preparar 20x solução salina tamponada com Tris (TBS) dissolvendo 122 g de Tris base e 180 g de cloreto de sódio em 850 mL de dH2O (pH 8,0). Ajuste o volume para 1 L com dH2O.</li>
	<li>Preparar o tampão de bloqueio dissolvendo 5 g de leite em pó desnatado com 1 mL de Tween-20 a 10%, 5 mL de 20x TBS e 94 mL de dH2O.</li>
	<li>Preparar tampão de anticorpos dissolvendo 3 g de BSA com 1 mL de Tween-20 a 10%, 5 mL de 20x TBS e 94 mL de dH2O.</li>
	<li>Preparar o tampão de lavagem misturando 100 mL de 20x TBS e 20 mL de Tween-20 10% (10x) com 1,88 L de dH2O.</li>
	<li>Preparar a solução reveladora misturando 1 volume de luminol/enhance com 1 volume de tampão de peróxido estável.</li>
</ol>2. Dissecção do músculo gastrocnêmio (GA) de camundongos15,16
<ol><li>Preparar tubos de 50 mL contendo 10 mL de PBS sobre gelo.</li>
	<li>Eutanásia dos camundongos em câmara de CO2 seguida de deslocamento cervical.</li>
	<li>Retire o músculo gastrocnêmio de ambas as pernas e transfira-os para o tubo com PBS no gelo. NOTA: Remova o músculo sóleo do músculo GA antes de transferir o músculo GA para PBS.</li>
</ol>3. Isolamento e cultura de fibroblastos primários em camundongos
<ol><li>Pese os músculos e transfira-os para um prato de 10 cm sob uma capa de biossegurança. Pique os músculos com bisturis até se tornar uma pasta fina.</li>
	<li>Transfira a pasta muscular finamente picada para um tubo de 50 mL e adicione 3,5x volume/mg de tecido de solução de digestão ao tubo e incube por 45 min a 37 °C. Misture bem a suspensão com uma pipeta de 5 mL a cada 10 min (isso ajudará na dissociação completa). NOTA: Alternativamente, um dissociador de tecido pode ser usado para esta etapa.</li>
	<li>Adicionar 20 mL de DMEM completo à suspensão celular para inativar a solução de digestão. Transfira para um filtro de células de nylon de 70 μm colocado em um tubo de 50 mL. Recolher o flow-through e lavar o filtro celular com mais 5 ml de DMEM completo.</li>
	<li>Centrifugar a suspensão celular a 300 x g à temperatura ambiente (TR) durante 10 minutos e remover cuidadosamente o sobrenadante.</li>
	<li>Ressuspender o pellet em 10 mL de DMEM completo e semear as células em uma placa de 10 cm.</li>
	<li>Cultivar as células a 37 °C, 5% CO 2, 3% O2 (passagem 0 ou P0). NOTA: 1) Essas culturas precisam ser mantidas em um baixo nível de oxigênio para garantir condições fisiológicas (o nível de O 2 no músculo esquelético está em torno de2,5%)17. 2) O número de passagem (por exemplo, P0) de uma cultura celular é um registro do número de vezes que a cultura foi subcultivada. 3) Fibroblastos primários em P0 são rotineiramente cultivados até 100% de confluência mais 1 dia (e somente para P0). Trata-se de purgar outros tipos de células e garantir que as células presentes na cultura sejam apenas fibroblastos, depletados de células miogênicas com base no exame microscópico. O músculo esquelético de um animal adulto aos 2 meses de idade contém cerca de 2% de progenitores miogênicos4. Além disso, os progenitores miogênicos geralmente não se ligam aos pratos não revestidos; portanto, perdem-se durante a subcultura18,19,20. As células miogênicas necessitam de um meio diferente (F10) suplementado com SFB a 20% e fator de crescimento básico de fibroblastos (bFGF)18. No DMEM completo, os fibroblastos médios apresentam maior taxa de proliferação que as células miogênicas, o que resulta na eliminação dessas células contaminantes antes da primeira passagem.</li>
	<li>Enxaguar as células P0 com PBS quando 100% confluente mais 1 dia. Adicionar 1 mL de solução de tripsinização e incubar a 37 °C, 5% CO 2, 3% O2 para separar as células. Pare a atividade enzimática usando 10 mL de DMEM completo.</li>
	<li>Centrifugar as células a 300 x g por 10 min em TR e ressuspender o pellet em 20 mL de DMEM completo e semear em uma placa de 15 cm (passagem 1 ou P1). As células são cultivadas até 80%-90% de confluência e podem ser expandidas até a passagem 3. NOTA: Dependendo dos experimentos a jusante a passagem 1, a passagem 2 (P2) ou a passagem 3 (P3) podem ser usadas.</li>
</ol>4. Semeadura das células e coleta do meio condicionado
<ol><li>Lavar os fibroblastos (P1, P2 ou P3) com 10 mL de PBS, adicionar 2,5 mL de solução de tripsinização às células e incubar a 37 °C, 5% CO 2, 3% O2. Pare a atividade enzimática usando 10 mL de DMEM completo. NOTA: Após a passagem 4, as células primárias são descartadas e não devem ser usadas para experimentos posteriores, pois mudam de característica.</li>
	<li>Recolher as células e centrifugar a 300 x g em RT durante 10 min.</li>
	<li>Ressuspendeu o pellet celular em 10 mL de meio livre de exossomo e contagem das células.</li>
	<li>Semeando as células a 1,5–2,0 x 106 células por prato (15 cm) e incubar a 37 °C, 5% CO 2, 3% O2.</li>
	<li>Coletar o meio condicionado entre 16 e 24 h em tubos de 50 mL sobre gelo. NOTA: Se as células não forem 80% confluentes, o meio livre exossomal fresco pode ser adicionado novamente e coletado após um período adicional de 16 a 24 horas. As duas coleções podem ser combinadas para processamento posterior.</li>
</ol>5. Purificação de exossomos utilizando diferencial e ultracentrifugação
NOTA: Todas as etapas são executadas a 4 °C ou no gelo. Equilibre o tubo com um tubo cheio de água quando necessário.
<ol><li>Centrifugar o meio condicionado a 300 x g por 10 min para remoção de células vivas e transferir o sobrenadante para um novo tubo de 50 mL.</li>
	<li>Remover as células mortas por centrifugação a 2.000 x g por 10 min e transferir o sobrenadante para um tubo de polipropileno de 38,5 mL.</li>
	<li>Centrifugar o sobrenadante a 10.000 x g por 30 min para a remoção de organelas, corpos apoptóticos e fragmentos de membrana. Transfira o sobrenadante para um tubo ultratransparente de 38,5 mL.</li>
	<li>Ultracentrifugar o sobrenadante a 100.000 x g por 1,5 h para pellet dos exossomos.</li>
	<li>Descarte cuidadosamente o sobrenadante, deixando aproximadamente 1 mL de meio condicionado, e lave o pellet do exossomo em um volume total de 30 mL de PBS gelado.</li>
	<li>Centrifugar a 100.000 x g por 1,5 h e descartar cuidadosamente o sobrenadante por pipetagem, tomando cuidado para não perturbar a pelota exossomal.</li>
	<li>Ressuspender o pellet em PBS gelado (~25 μL por placa de 15 cm) e medir a concentração de proteína usando o kit de ensaio de proteína BCA e um leitor de microplacas a 562 nm. NOTA: A concentração de proteínas é medida por uma diluição de 1:2 com 0,1% de Triton-X100 em dH2O. O rendimento de exossomos de uma placa de 15 cm (20 mL de meio condicionado) de fibroblastos primários a 80% de confluência é de ~3–4 μg.</li>
</ol>6. Caracterização de exossomos pelo gradiente de densidade de sacarose
<ol><li>Coloque o gradiente de sacarose em um tubo ultratransparente de 13 mL pipetando (de baixo para cima) o seguinte: 1,5 mL de soluções de sacarose de 2,0 M, 2,5 mL de 1,3 M, 2,5 mL de 1,16 M, 2,0 mL de 0,8 M e 2,0 mL de soluções de sacarose 0,5 M.</li>
	<li>Misturar 30–100 μg de exossomos com solução de sacarose 0,25 M e ajustar o volume para 1 mL.</li>
	<li>Carregar cuidadosamente os exossomos sobre os gradientes e ultracentrifugar as amostras por 2,5 h a 100.000 x g e 4 °C.</li>
	<li>Retire os tubos da ultracentrífuga e coloque-os sobre gelo e colete frações de 1,0 mL a partir do topo do gradiente. Transfira-os para tubos de 1,7 mL sobre gelo.</li>
	<li>Adicione 110 μL de 100% de TCA a cada tubo, misture bem e incube por 10 min no TR.</li>
	<li>Centrifugar por 10 min a 10.000 x g e RT e descartar cuidadosamente o sobrenadante.</li>
	<li>Ressuspender o pellet em 500 μL de etanol 80% pré-resfriado e deixar as amostras lavarem por 10 min a -20 °C.</li>
	<li>Centrifugar por 10 min a 10.000 x g e 4 °C e descartar o sobrenadante.</li>
	<li>Secar o pellet por 10–15 min em RT e ressuspendê-lo em PBS para experimentos in vitro. Medir a concentração de proteína usando o kit de ensaio de proteína BCA ou ressuspender o pellet diretamente em 14,5 μL de dH2O, 5 μL de tampão Laemmli 4x e 0,5 μL de TDT 1 M para análises de western blot.</li>
</ol>7. Detecção de exossomos por análise de western blot
<ol><li>Misturar 5–10 μg de exossomas do passo 5.7 com 5 μL de tampão Laemmli 4x e 0,5 μL de TDT 1 M e, em seguida, ajustar o volume final para 20 μL com dH2O. Aquecer todas as amostras, incluindo as do passo 6.9, durante 5 minutos a 98 °C.</li>
	<li>Coloque as amostras em um gel sem coloração TGX a 10% e carregue as escadas de proteína de acordo com as recomendações do fabricante. OBS: Escolha a porcentagem do gel de acordo com o peso molecular da proteína de interesse.</li>
	<li>Passe o gel a 100 V por ~1 h em tampão de corrida até que o corante de carga esteja na parte inferior do gel. OBS: O tempo de execução pode variar de acordo com o equipamento utilizado ou tipo e porcentagem de gel.</li>
	<li>Imagem do gel. Imagens dos géis livres de manchas são usadas como controle de carga para immunoblots.</li>
	<li>Transfira o gel usando uma membrana de PVDF por 1,5 h a 80 V ou durante a noite a 30 V a 4 °C.</li>
	<li>Bloquear as membranas em tampão de bloqueio por 1 h na RT.</li>
	<li>Incubar as membranas para anticorpos específicos (Tabela 2) em tampão de anticorpos durante a noite a 4 °C com agitação.</li>
	<li>Lavar as membranas em tampão de lavagem e incubá-las com os anticorpos secundários apropriados (Tabela 2) por 1 h na RT com agitação.</li>
	<li>Lave as membranas no tampão de lavagem e visualize as membranas usando a solução revelação e um imageador baseado em câmera. Alternativamente, use um filme de raios-X para desenvolver as membranas.</li>
</ol>

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Nenhum dos autores tem conflitos de interesse a declarar.

Um passo crítico para o sucesso do isolamento dos exossomos dos meios de cultura, conforme descrito neste protocolo, é o estabelecimento e a manutenção adequados de culturas primárias de fibroblastos de camundongos do músculo esquelético adulto. Essas culturas precisam ser mantidas em um baixo nível de oxigênio para garantir condições fisiológicas (o nível de O2 no músculo esquelético é de ~2,5%)15. Os fibroblastos primários mudam de características quando passados em ...

Exossomos são vesículas extracelulares heterogêneas que variam em tamanho de 30 a 150 nm. Estabelecem-se como peças-chave nos processos fisiológicos e patológicos, dada a sua distribuição ubíqua em tecidos e órgãos 1,2. Os exossomos carregam uma carga complexa de proteínas, lipídios, tipos de DNA e tipos de RNA, que variam de acordo com o tipo de células das quais são derivadas 1,2,3. Os exossomos são enriquecidos em proteínas que têm diferentes funções (ou seja, tetraspaninas, incluindo CD9 e CD63) são responsáveis por eventos de fusão. Por exemplo, as proteínas de choque térmico HSP70 e HSP90 estão envolvidas na ligação e apresentação de antígenos. Adicionalmente, Alix, Tsg101 e flotino, participam da biogênese e liberação do exossomo e são amplamente utilizados como marcadores dessas nanovesículas2,3,4.
Os exossomos também contêm uma variedade de RNAs (isto é, microRNAs, RNAs longos não codificantes, RNAs ribossomais) que podem ser transferidos para as células receptoras, onde influenciam a sinalização a jusante3. Por ser delimitada por uma única unidade de membrana, a bioatividade do exossomo depende não apenas da carga de proteínas e ácidos nucléicos, mas também dos componentes lipídicos da membrana limitante1. As membranas exossômicas são enriquecidas com fosfatidilserina, ácido fosfatídico, colesterol, esfingomielina, ácido araquidônico e outros ácidos graxos, os quais podem influenciar a estabilidade do exossomo e a topologia da membrana 2,3. Como resultado do arranjo de carga e lipídios, os exossomos iniciam vias de sinalização nas células receptoras e participam da manutenção da fisiologia tecidual normal 1,2,4,5. Em determinadas condições patológicas (neurodegeneração, fibrose e câncer), eles têm demonstrado desencadear e propagar estímulos patológicos 4,6,7,8,9,10,11.
Devido à sua capacidade de propagar sinais para locais vizinhos ou distantes, os exossomos tornaram-se valiosos biomarcadores para o diagnóstico ou prognóstico de condições de doença. Além disso, exossomos têm sido utilizados experimentalmente como veículos de compostos terapêuticos 2,12. O potencial de aplicação dessas nanovesículas na clínica torna o método de isolamento cada vez mais importante para atingir o máximo rendimento, pureza e reprodutibilidade. Diferentes técnicas para o isolamento de exossomos têm sido desenvolvidas e implementadas. Geralmente, os exossomos podem ser isolados de meios de cultura celular condicionados ou fluidos corporais por centrifugação diferencial, cromatografia de exclusão de tamanho e captura imunológica (usando kits disponíveis comercialmente). Cada abordagem tem vantagens e desvantagens únicas que foram discutidas anteriormente 1,2,13,14.
O protocolo delineado enfoca 1) isolamento e cultura de fibroblastos primários do músculo gastrocnêmio de camundongos adultos e 2) purificação e caracterização de exossomos liberados no meio de cultura por essas células. Atualmente, não existe um protocolo bem estabelecido para o isolamento de exossomos de fibroblastos primários para estudos funcionais. Os fibroblastos primários não secretam grandes quantidades de exossomos, tornando o processo de isolamento e purificação desafiador. Este protocolo descreve a purificação de grandes quantidades de exossomos puros de grandes volumes de cultura, mantendo sua integridade morfológica e atividade funcional. Exossomos purificados obtidos de meio condicionado têm sido usados com sucesso em experimentos de captação in vitro para induzir vias de sinalização específicas em células receptoras. Eles também têm sido utilizados para análises proteômicas comparativas de cargas exossômicas de múltiplas amostrasbiológicas 4.

<table>
	<tbody>
		<tr>
			<td>10 cm dishes</td>
			<td>Midwest Scientific, TPP</td>
			<td>TP93100</td>
			<td></td>
		</tr>
		<tr>
			<td>15 cm dishes</td>
			<td>Midwest Scientific</td>
			<td>TP93150</td>
			<td></td>
		</tr>
		<tr>
			<td>BCA protein assay kit</td>
			<td>Thermo Fisher Scientific, Pierce</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin Fraction V</td>
			<td>Roche</td>
			<td>10735094001</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C1016-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5430R with rotors FA-35-6-30/ FA-45-48-11</td>
			<td>Eppendorf</td>
			<td>022620659/5427754008</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemidoc MP imaging system</td>
			<td>BioRad</td>
			<td>12003154</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase P</td>
			<td>Sigma, Roche</td>
			<td>11 213 857 001</td>
			<td>100 mg</td>
		</tr>
		<tr>
			<td>cOmplete protease inhibitor cocktail</td>
			<td>Millipore/Sigma, Roche</td>
			<td>11697498001</td>
			<td></td>
		</tr>
		<tr>
			<td>Criterion Blotter with plate electrodes</td>
			<td>BioRad</td>
			<td>1704070</td>
			<td></td>
		</tr>
		<tr>
			<td>Criterion TGX stain-free protein gel</td>
			<td>BioRad</td>
			<td>5678034</td>
			<td>10% 18-well, midi-gel</td>
		</tr>
		<tr>
			<td>Criterion vertical electrophoresis cell (midi)</td>
			<td>BioRad</td>
			<td>NC0165100</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Sigma, Roche</td>
			<td>04 942 078 001</td>
			<td>neutral protease, grade II</td>
		</tr>
		<tr>
			<td>Dithiothreitol</td>
			<td>Sigma/Millipore, Roche</td>
			<td>10708984001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modification Eagles Medium</td>
			<td>Corning</td>
			<td>15-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 200 proof</td>
			<td>Pharmco by Greenfield Global</td>
			<td>111000200</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 50 mL conical centrifuge tubes</td>
			<td>Corning</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluostar Omega multi-mode microplate reader</td>
			<td>BMG Labtech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX supplement</td>
			<td>Thermo Fisher Scientific, Gibco</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Fisher Scientific</td>
			<td>A144S-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Immobilon-P Transfer membranes</td>
			<td>Millipore</td>
			<td>IPVH00010</td>
			<td></td>
		</tr>
		<tr>
			<td>Laemmli sample buffer (4x)</td>
			<td>BioRad</td>
			<td>1610747</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium acetate solution</td>
			<td>Sigma</td>
			<td>63052-100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-fat dry milk</td>
			<td>LabScientific</td>
			<td>M-0842</td>
			<td></td>
		</tr>
		<tr>
			<td>O2/CO2 incubator</td>
			<td>Sanyo</td>
			<td>MC0-18M</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Fisher Scientific, Gibco</td>
			<td>15140-122</td>
			<td>10,000 U/ml</td>
		</tr>
		<tr>
			<td>Premium microcentrifuge tubes</td>
			<td>Fisher Scientific, Midwest Scientific</td>
			<td>AVSC1510</td>
			<td>1.7 mL</td>
		</tr>
		<tr>
			<td>Protected disposable scalpels</td>
			<td>Fisher Scientific, Aspen Surgical Bard-Parker</td>
			<td>372610</td>
			<td></td>
		</tr>
		<tr>
			<td>Running buffer</td>
			<td>BioRad</td>
			<td>1610732</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific, Fisher Chemical</td>
			<td>S271-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Stericup Quick release-GP sterile vacuum filtration system</td>
			<td>Millipore</td>
			<td>S2GPU05RE</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Sterile cell strainer (70 mm)</td>
			<td>Fisher Scientific, Fisher brand</td>
			<td>22-363-548</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher Scientific, Fisher Chemical</td>
			<td>S5-500</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperSignal west Femto</td>
			<td>Thermo Fisher Scientific</td>
			<td>34096</td>
			<td></td>
		</tr>
		<tr>
			<td>Thin wall Polypropylene tubes</td>
			<td>Beckman Coulter</td>
			<td>326823</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer buffer</td>
			<td>BioRad</td>
			<td>16110734</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichloroacetic Acid</td>
			<td>Sigma</td>
			<td>91228-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>BioRad</td>
			<td>1610719</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X100 solution</td>
			<td>Sigma</td>
			<td>93443-100mL</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express Enzyme</td>
			<td>Thermo Fisher Scientific, Gibco</td>
			<td>12604-013</td>
			<td>No phenol red</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>BioRad</td>
			<td>#1610781</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-centrifuge Optima XPM</td>
			<td>Beckman Coulter</td>
			<td>A99842</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-clear tube (14x89 mm)</td>
			<td>Beckman Coulter</td>
			<td>344059</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-clear tubes (25x89 mm)</td>
			<td>Beckman Coulter</td>
			<td>344058</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath Isotemp 220</td>
			<td>Fisher Scientific</td>
			<td>FS220</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and characterization of exosomes from skeletal muscle fibroblasts

Exossomos são pequenas vesículas extracelulares liberadas por praticamente todas as células e secretadas em todos os fluidos biológicos. Muitos métodos foram desenvolvidos para o isolamento dessas vesículas, incluindo ultracentrifugação, ultrafiltração e cromatografia de exclusão de tamanho. No entanto, nem todos são adequados para purificação e caracterização de exossomos em larga escala. Delinea-se aqui um protocolo para o estabelecimento de culturas de fibroblastos primários isolados de músculos esqueléticos adultos de camundongos, seguido de purificação e caracterização de exossomos dos meios de cultura dessas células. O método baseia-se no uso de etapas de centrifugação sequencial seguidas de gradientes de densidade de sacarose. A pureza das preparações exossômicas é então validada por análises de western blot usando uma bateria de marcadores canônicos (i.e., Alix, CD9 e CD81). O protocolo descreve como isolar e concentrar exossomos bioativos para microscopia eletrônica, espectrometria de massa e experimentos de captação para estudos funcionais. Ele pode ser facilmente ampliado para cima ou para baixo e adaptado para isolamento de exossomos de diferentes tipos de células, tecidos e fluidos biológicos.

Este protocolo é adequado para a purificação de exossomos de grandes volumes de meio condicionado de forma custo-efetiva. O procedimento é altamente reprodutível e consistente. A Figura 1 mostra a imagem de microscopia eletrônica de transmissão (MET) de exossomos purificados do meio de cultura de fibroblastos primários de camundongos. A Figura 2 mostra o padrão de expressão proteica dos marcadores exossômicos canônicos e a ausência de contaminantes ...

Watch this Scientific Journal Video about Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts at JoVE.com

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Este protocolo ilustra 1) o isolamento e cultura de fibroblastos primários do músculo gastrocnêmio de camundongos adultos, bem como 2) purificação e caracterização de exossomos usando um método de ultracentrifugação diferencial combinado com gradientes de densidade de sacarose seguidos por análises de western blot.

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 ...

isolation-characterization-exosomes-from-skeletal-muscle

Research

JoVE Journal

Biology

3.2K visualizações.  St. Jude Children's Research Hospital. Este protocolo ilustra 1) o isolamento e cultura de fibroblastos primários do músculo gastrocnêmio de camundongos adultos, bem como 2) purificação e caracterização de exossomos usando um método de ultracentrifugação diferencial combinado com gradientes de densidade de sacarose seguidos por análises de western blot.

Vídeo: Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

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 ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

Purificação de Progenitores do músculo esquelético

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

Isolation and Characterization of RNA-Containing Exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes1, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane2. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk3-6. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen7,8, eradication of established tumors in mice9, inhibition and activation of natural killer cells10-12, promotion of differentiation into T regulatory cells13, stimulation of T cell proliferation14 and induction of T cell apoptosis15. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA16. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA17. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer.

This paper demonstrates methods for the isolation, purification and detection of exosomes, as well as techniques for analysis of their molecular content. These methods are adaptable for exosome isolation from both cell culture media and biological fluids, and can beyond analysis of molecular content also be useful in functional studies.

Isolamento e Caracterização de RNA-Contendo exossomos

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of ...

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 of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Isolamento de mitocôndrias de quantidades mínimas de Rato Músculo Esquelético para medições High Throughput Microplate respiratórias

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

Isolation of Intact Mitochondria from Skeletal Muscle by Differential Centrifugation for High-resolution Respirometry Measurements

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential centrifugation at low- and high-speed is commonly used to isolate mitochondria from tissues and cultured cells. Crude mitochondrial fractions obtained by differential centrifugation are used for respirometry measurements. The differential centrifugation technique is based on the separation of organelles according to their size and sedimentation velocity. The isolation of mitochondria is performed immediately after tissue harvesting. The tissue is immersed in an ice-cold homogenization medium, minced using scissors and homogenized in a glass homogenizer with a loose-fitting pestle. The differential centrifugation technique is efficient, fast and inexpensive and the mitochondria obtained by differential centrifugation are pure enough for respirometry assays. Some of the limitations and disadvantages of isolated mitochondria, based on differential centrifugation, are that the mitochondria can be damaged during the homogenization and isolation procedure and that large amounts of the tissue biopsy or cultured cells are required for the mitochondrial isolation.

Here, a quadriceps muscle specimen is taken from an anaesthetized pig and mitochondria are isolated by differential centrifugation. Then, the respiratory rates of mitochondrial respiratory chain complexes I, II and IV are determined using high-resolution respirometry.

Isolamento de mitocôndrias intactas de Músculo Esquelético por centrifugação diferencial para medições Respirometria de alta resolução

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential ...

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that mitigate fibrosis. However, fibroblast research has been hindered due to the lack of universally acceptable fibroblast markers to identify quiescent as well as activated fibroblasts. Fibroblasts are a heterogenous cell population, making them difficult to isolate and characterize. The presented protocol describes three different methods to enrich fibroblasts and myofibroblasts from uninjured and injured mouse hearts. Using a standard and reliable protocol to isolate fibroblasts will enable the study of their roles in homeostasis as well as fibrosis modulation.

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

Isolamento e Caracterização de Fibroblastos Cardíacos Adultos e Miofibroblastos

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...

Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly isolated by coronary perfusion with digestive enzymes. However, isolating human cardiomyocytes is challenging because human myocardial specimens usually do not allow for coronary perfusion, and alternative isolation protocols result in poor yields of viable cells. In addition, human myocardial specimens are rare and only regularly available at institutions with on-site cardiac surgery. This hampers the translation of findings from animal to human cardiomyocytes. Described here is a reliable protocol that enables efficient isolation of ventricular myocytes from human and animal myocardium. To increase the surface-to-volume ratio while minimizing cell damage, myocardial tissue slices 300 &#181;m thick are generated from myocardial specimens with a vibratome. Tissue slices are then digested with protease and collagenase. Rat myocardium was used to establish the protocol and quantify yields of viable, calcium-tolerant myocytes by flow-cytometric cell counting. Comparison with the commonly used tissue-chunk method showed significantly higher yields of rod-shaped cardiomyocytes (41.5 &#177; 11.9 vs. 7.89 &#177; 3.6%, p &#60; 0.05). The protocol was translated to failing and non-failing human myocardium, where yields were similar as in rat myocardium and, again, markedly higher than with the tissue-chunk method (45.0 &#177; 15.0 vs. 6.87 &#177; 5.23 cells/mg, p &#60; 0.05). Notably, with the protocol presented it is possible to isolate reasonable numbers of viable human cardiomyocytes (9&#8211;200 cells/mg) from minimal amounts of tissue (&#60;50 mg). Thus, the method is applicable to healthy and failing myocardium from both human and animal hearts. Furthermore, it is possible to isolate excitable and contractile myocytes from human tissue specimens stored for up to 36 h in cold cardioplegic solution, rendering the method particularly useful for laboratories at institutions without on-site cardiac surgery.

Presented is a protocol for the isolation of human and animal ventricular cardiomyocytes from vibratome-cut myocardial slices. High yields of calcium-tolerant cells (up to 200 cells/mg) can be obtained from small amounts of tissue (&#60;50 mg). The protocol is applicable to myocardium exposed to cold ischemia for up to 36 h.

Isolamento de Cardiomiócitos Ventriculares Humanos de fatias de miocárdio cortadas por vibratome

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly ...