Agradecemos ao Dr. Yitzchak Oschry e Dr. Orit Sagi-Asif por sua ajuda com a classificação FACS. Esta pesquisa foi suportada por concessões para a NE do Sétimo Programa-Quadro da União Europeia (FP7/2007-2013) sob subvenção n ° [276890], a partir da Israel Cancer Association (# 20110078), e da Israel Cancer Research Fund (Investigação Prémio Carreira de Desenvolvimento).

1. Dissecando tecido mamário ou pele de camundongos <ol><li> Antes de dissecação do tecido a partir de ratinhos desejado, preparar os seguintes materiais e reagentes: Suprimentos: <ul><li> Ferramentas de operação (lavado com detergente e, em seguida mergulhados em etanol a 70%). </li><li> Superfície de isopor para descansar o mouse sobre durante a dissecção. </li><li> Pinos ou agulhas para manter ratos durante a dissecção. </li><li> Frasco de vidro com tampa para a digestão, contendo uma barra magnética (autoclavado). </li><li> Banho de água a 37 ° C, contendo um agitador magnético submersível e água suficiente para submergir o frasco de digestão, enquanto repousa sobre a placa de agitação. </li></ul> Reagentes: <ul><li> Tampão FACS I: (4 ° C): Fosfato de Dulbecco Buffered Saline CMF (Cálcio Magnésio livre) + BSA a 0,5% </li><li> Colagenase solução (feita imediatamente antes da utilização): 0,05 g de colagenase II 0,05 g de colagenase IV 0,01 g desoxirribonuclease 20ml de tampão de FACS I. Manter solução de colagenase em gelo até à utilização. </li><li> PharmLyse (amônio Cloreto de Lise). </li><li> Tampão FACS II: Fosfato de Dulbecco Buffered Saline CMF + FCS a 1%. </li></ul></li><li> Dissecção das glândulas mamárias (ver também 17). <ul><li> Eutanásia ratos fêmeas usando inalação de dióxido de carbono (CO 2). </li><li> Em uma superfície de isopor, coloque o mouse sobre a sua volta e fixar firmemente todos os quatro membros, usando agulhas de calibre 25 (Figura 1A). Pulverizar o mouse generosamente com etanol 70%. </li><li> Utilizando uma pinça, puxe a pele abdominal na linha média e fazer uma pequena incisão com tesoura afiada. </li><li> A partir da incisão, cortar a pele até ao pescoço do animal, evitando a punção da cavidade abdominal ou torácica. </li><li> Cortar a pele das patas traseiras a partir da incisão na linha média para a perna, o que resulta numa &quot;forma de Y&quot;. </li><li> Puxe a pele longe do corpo do mouse e definir, expondoas glândulas mamárias acoplada à parte inferior da pele (Figura 1B). </li><li> Use Q-dicas para gentilmente separar glândulas mamárias da pele durante o corte do tecido conjuntivo com pequenas tesouras afiadas para separar a glândula mamária para a coluna do mouse. </li><li> As glândulas abdominais (# 4, 5) são os melhores para este protocolo. Também é possível utilizar a glândula mamaria 2 º, mas ter em mente que estas glândulas são anatomicamente localizado na proximidade do músculo peitoral que podem ser difíceis de separar, o que resulta em células de origem mista do tecido. </li><li> Nota: Quando dissecar tecido tumoral, recomenda-se para remover as regiões necróticas. </li></ul></li><li> A dissecção do tecido da pele: A maneira mais fácil de obter tecido da pele, sem ter que lidar com a depilação é usar orelhas de rato. Se uma quantidade maior de tecido é necessário, você pode cortar o cabelo de torso mouse e dissecar pele. <ul><li> Euthanize ratos usando inalação de CO2. </li><li> Utilizando uma tesoura afiada e cortar ambas as orelhas e colocar imediatamente no frasco de digestão contendo um pequeno volume (~ 0,5 ml) de PBS em gelo. </li></ul></li></ol> 2. Preparação de suspensão de células da glândula mamária / pele Único <ul><li> Coloque glândulas mamárias / pele no frasco de digestão contendo um pequeno volume de PBS em gelo. Se o tecido é sangrenta x2 lavagem com PBS e, em seguida, descartar excesso de PBS. </li><li> Picar cuidadosamente com tesoura curva. </li><li> Adicionar (nota: esta quantidade de solução de colagenase será eficiente dissociar aproximadamente 5 g de tecido mamário ou tumor e orelhas de até 15 ratos) 20 ml de solução de colagenase. </li><li> Colocar imediatamente na placa de agitar a 37 ° C banho-maria, e incubar durante 15 min, sob agitação a uma velocidade média. </li></ul> Nota: Os tempos de incubação ideais podem variar se digerir outros tecidos. <ul><li> Para parar a reacção, adicionar 30 ml de DMEM frio + 10% FCS. </li><li> Coe o throug mistura de tecido / mídiaha 70 uM coador célula colocado no topo de um tubo cónico de 50 ml. </li><li> Centrifugar o tubo cónico de 5 min a 450 xg a 4 ° C. </li></ul> 3. Red Blood Cells Lise Nota: este passo não é necessário se os ratos estavam de coração perfundidos com PBS antes de serem sacrificadas. <ul><li> Aspirar o sobrenadante com uma pipeta de vidro ligado a vácuo, sem perturbar o sedimento celular. </li><li> Para lisar RBC, ressuspender o pellet em 10 células solução PharmLyse ml e incubar durante 5 min à temperatura ambiente. </li><li> Neutraliza-se a solução PharmLyse por adição de cerca de 40 ml de tampão FACS I. </li><li> Girar o tubo cónico com as células durante 5 min a 450 xg a 4 ° C. </li><li> Aspirar o sobrenadante. </li></ul> 4. Bloqueio de Fc Endógeno <ul><li> Opcional: células de contagem para determinar o volume apropriado para os passos seguintes. </li><li> Ressuspender as células em 1-3 ml de tampão de FACS (dependendo o In número de células e quantidade de anticorpos que deseja usar). </li><li> Adicionar FcBlock (anti-rato de CD16/CD32) para chegar a diluição 1:50 (10 ug / ml), e incubar em gelo durante 10-20 &#39;. Não há necessidade de lavar FcBlock. </li></ul> 5. Coloração <ul><li> Tirar alíquotas de 100 ul a tubos eppendorf para ser utilizado como um controlo não-coradas e os controlos de cor individuais (de acordo com o número de anticorpos fluorescentes utilizadas). </li><li> Para fibroblastos etiqueta: adicionar anti-PDGFRα (directamente conjugado com fluoróforo) para uma diluição de 1:50 (10 ug / ml). </li><li> Para etiquetar outras populações de células (por exemplo células do sistema imune, macrófagos, células endoteliais, etc): adicionar apropriadas anticorpos conjugados com fluorescência para marcadores de superfície, para uma diluição de 1:50. </li></ul> Nota: embora PDGFRα é um marcador robusto para fibroblastos, recomenda-se a incluir anticorpos para as células do sistema imunológico e para as células epiteliais, a fim de excluir qualquer positivo duplopopulações e, assim, melhorar a pureza de fibroblastos isolados. <ul><li> Adicionam-se 2 jil de cada um dos anticorpos para cada uma das cores individuais tubos eppendorf de controlo. </li><li> Incubar as células com anticorpos para 1 hora em gelo, no escuro. </li><li> Para lavar: encher os tubos com tampão FACS I e girar durante 5 minutos a 450 xg a 4 ° C. </li><li> Aspirar o sobrenadante, Ressuspender as células em tampão de SCAF I a uma concentração de aproximadamente 2 x 10 6 células / ml, ou qualquer que seja a concentração mais adequada para a sua classificação com classificador FACS disponível. </li></ul> Opcional: As células podem ser ressuspendidas em tampão FACS II durante a triagem. Isto pode melhorar a viabilidade celular. No entanto, a exposição a factores de soro podem ter um efeito de expressão do gene, o que deve ser testado, e tomados em consideração. <ul><li> Transferir células marcadas em tubos de FACS com células: filtro de topo do filtro no tubo para evitar grumos celulares. </li><li> Para excluir as células mortas, adicionar 7AAD (0,5 ug / ml) ou Propidium Iodeto (PI) para amostras (exceto para o controle de não-coradas). </li></ul> Nota: Para evitar o desperdício de células, você pode adicionar 7AAD/PI à amostra não-manchado após a gravação nos FACS, e usar de novo como 7AAD somente controle. <ul><li> Manter as amostras em gelo ao analisar. </li></ul> 6. Isolamento de células por FACS (Nota: esta parte do protocolo requer conhecimento prévio em funcionamento de um separador de FACS, por exemplo, BD FACS AriaII, ou a assistência de um técnico especializado). <ul><li> Usando o software classificador FACS (BD FACSDiva), preparar lotes de análise adequados para analisar dispersão frontal (FSC), dispersão lateral (SSC), 7AAD, FITC, PE e qualquer outro fluoróforo usado para células de etiquetas (Figura 2). </li><li> Antes de carregar cada amostra para o classificador de celular, brevemente vortex para ressuspender as células. </li><li> Comece analisando a amostra sem mácula, a fim de definir a propagação para a população total de células, para deixar de fora celdetritos l, e para determinar a autofluorescência ou coloração de fundo. </li><li> Analisar amostras não coradas + 7AAD para determinar e porta a população de células vivas a partir da população total de células. </li><li> Analise cada controle de cor única para determinar e calibrar parâmetros, tais como a compensação. </li><li> Analisar a amostra corada para definir a posição das portas para classificação. </li><li> Uma vez que os parâmetros e as portas para as populações de células pretendidas tenham sido definidos, começam classificando as populações de células marcadas em tubos de Eppendorf ou tubos cónicos de 15 ml contendo meio de cultura ou de ARN de tampão de lise (como RLT tampão ou Trizol). </li></ul> Nota: Se a classificação de um grande número de células, isso não é recomendado para ordenar directamente em tampão de lise de ARN, tal como o grande volume de fluido de revestimento que acompanha as células irão diluir o tampão de lise e reduz o rendimento. <ul><li> Girar células-se imediatamente após a classificação está terminado, e transferência para meio fresco ou tampão de lise RNA, dependente sobre o propósito da sua experiência. </li><li> Se a classificação, a fim de células de cultura isoladas, executar tipo estéril. Para uma melhor recuperação de células, use vermelho de fenol livre DMEM + FCS a 5%, em vez de tampão FACS II e recolher as células em meio de cultura com FCS 20%. </li><li> Se cultura ordenadas fibroblastos, alwaysplate em placas de colágeno revestidos e nunca diretamente no plástico como isso resulta em ativação de fibroblastos levando a mudanças em sua expressão gênica. </li></ul>

<ol>
	<li>Kalluri, R., Zeisberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblasts+in+cancer.">Fibroblasts in cancer.</a> Nat. Rev. Cancer. 6, 392-401 (2006).</li><li>Shimoda, M., Mellody, K. T., Orimo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carcinoma-associated+fibroblasts+are+a+rate-limiting+determinant+for+tumour+progression.">Carcinoma-associated fibroblasts are a rate-limiting determinant for tumour progression.</a> Seminars in cell &amp; developmental biology. 21, 19-25 (2010).</li><li>Orimo, A., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+stromal+fibroblasts+in+tumors.">Heterogeneity of stromal fibroblasts in tumors.</a> Cancer Biol. Ther. 6, 618-619 (2007).</li><li>Ostman, A., Augsten, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer-associated+fibroblasts+and+tumor+growth--bystanders+turning+into+key+players.">Cancer-associated fibroblasts and tumor growth--bystanders turning into key players.</a> Current opinion in genetics &amp; development. 19, 67-73 (2009).</li><li>Allinen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characterization+of+the+tumor+microenvironment+in+breast+cancer.">Molecular characterization of the tumor microenvironment in breast cancer.</a> Cancer Cell. 6, 17-32 (2004).</li><li>Bhowmick, N. A., Neilson, E. G., Moses, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+fibroblasts+in+cancer+initiation+and+progression.">Stromal fibroblasts in cancer initiation and progression.</a> Nature. 432, 332-337 (2004).</li><li>Orimo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+fibroblasts+present+in+invasive+human+breast+carcinomas+promote+tumor+growth+and+angiogenesis+through+elevated+SDF-1/CXCL12+secretion.">Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion.</a> Cell. 121, 335-348 (2005).</li><li>Pietras, K., Ostman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallmarks+of+cancer:+interactions+with+the+tumor+stroma.">Hallmarks of cancer: interactions with the tumor stroma.</a> Experimental cell research. 316, 1324-1331 (2010).</li><li>Erez, N., Truitt, M., Olson, P., Arron, S. T., Hanahan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer-Associated+Fibroblasts+Are+Activated+in+Incipient+Neoplasia+to+Orchestrate+Tumor-Promoting+Inflammation+in+an+NF-kappaB-Dependent+Manner.">Cancer-Associated Fibroblasts Are Activated in Incipient Neoplasia to Orchestrate Tumor-Promoting Inflammation in an NF-kappaB-Dependent Manner.</a> Cancer Cell. 17, 135-147 (2010).</li><li>Sugimoto, H., Mundel, T. M., Kieran, M. W., Kalluri, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+fibroblast+heterogeneity+in+the+tumor+microenvironment.">Identification of fibroblast heterogeneity in the tumor microenvironment.</a> Cancer Biol. Ther. 5, 1640-1646 (2006).</li><li>Neumann, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+culture+and+passaging+alters+gene+expression+pattern+and+proliferation+rate+in+rheumatoid+arthritis+synovial+fibroblasts.">Cell culture and passaging alters gene expression pattern and proliferation rate in rheumatoid arthritis synovial fibroblasts.</a> Arthritis research &amp; therapy. 12, R83 .</li><li>Sherr, C. J., DePinho, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+senescence:+mitotic+clock+or+culture+shock.">Cellular senescence: mitotic clock or culture shock.</a> Cell. 102, 407-410 (2000).</li><li>DeNardo, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+complexity+in+breast+cancer+predicts+overall+survival+and+functionally+regulates+response+to+chemotherapy.">Leukocyte complexity in breast cancer predicts overall survival and functionally regulates response to chemotherapy.</a> Cancer Discovery. 1, 54-67 (2011).</li><li>Pahler, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+in+tumor-promoting+inflammation:+impairment+of+macrophage+recruitment+evokes+a+compensatory+neutrophil+response.">Plasticity in tumor-promoting inflammation: impairment of macrophage recruitment evokes a compensatory neutrophil response.</a> Neoplasia. 10, 329-340 (2008).</li><li>Pietras, K., Pahler, J., Bergers, G., Hanahan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functions+of+paracrine+PDGF+signaling+in+the+proangiogenic+tumor+stroma+revealed+by+pharmacological+targeting.">Functions of paracrine PDGF signaling in the proangiogenic tumor stroma revealed by pharmacological targeting.</a> PLoS Med. 5, e19 (2008).</li><li>Quante, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow-derived+myofibroblasts+contribute+to+the+mesenchymal+stem+cell+niche+and+promote+tumor+growth.">Bone marrow-derived myofibroblasts contribute to the mesenchymal stem cell niche and promote tumor growth.</a> Cancer Cell. 19, 257-272 (2011).</li><li>Plante, I., Stewart, M. K., Laird, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+Mammary+Gland+Development+and+Function+in+Mouse+Models.">Evaluation of Mammary Gland Development and Function in Mouse Models.</a> J. Vis. Exp. (53), e2828 (2011).</li><li>Xouri, G., Christian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+and+function+of+tumor+stroma+fibroblasts.">Origin and function of tumor stroma fibroblasts.</a> Seminars in cell &amp; developmental biology. 21, 40-46 (2010).</li></ol>

Não há conflitos de interesse declarados.

Embora experiências efectuadas em cultura de tecidos podem ser informativas e sugerem princípios funcionais que podem ser verificadas in vivo, que se sabe que ocorrem grandes mudanças na expressão de genes de células em cultura 11,12. A fim de evitar uma etapa de cultura de tecido, quando o perfil de expressão de genes em fibroblastos, foi desenvolvido um protocolo que permite o isolamento dos normais, bem como as associadas câncer fibroblastos de rato fresco ou de tecido humano. Este protocol...

<table border="1"&gt;<tbody&gt;<tr&gt;<td<strong&gt; Nome do reagente</strong</td&gt;<td<strong&gt; Empresa</strong</td&gt;<td<strong&gt; Número de catálogo</strong</td&gt;<td<strong&gt; Comentários (opcional)</strong</td&gt;</tr&gt;<tr&gt;<td&gt; DMEM</td&gt;<td&gt; Gibco</td&gt;<td&gt; 41965</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; PBS</td&gt;<td&gt; Biological Industries</td&gt;<td&gt; 02-023-1A</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Colagenase II</td&gt;<td&gt; Worthington</td&gt;<td&gt; LS4176</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Colagenase IV</td&gt;<td&gt; Worthington</td&gt;<td&gt; LS4188</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Desoxirribonuclease</td&gt;<td&gt; Worthington</td&gt;<td&gt; LS2007</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; PharmLyse</td&gt;<td&gt; BD</td&gt;<td&gt; 555899</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; 70 um filtro celular</td&gt;<td&gt; SPL</td&gt;<td&gt; 93070</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Purificada anti-rato CD16/CD32</td&gt;<td&gt; BD Pharmingen</td&gt;<td&gt; 553142</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Via sonda (7AAD)</td&gt;<td&gt; E-Bioscience</td&gt;<td&gt; 00-6993-50</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Anti-rato CD140a-PE (PDGFRA)</td&gt;<td&gt; E-Bioscience</td&gt;<td&gt; 12-1401-81</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Anti-mouse FITC F4/80-</td&gt;<td&gt; Cederlane</td&gt;<td&gt; CL8940F</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; DMEM w / o Vermelho de Fenol</td&gt;<td&gt; Gibco</td&gt;<td&gt; 31053</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Colágeno tipo I</td&gt;<td&gt; BD Biosciences</td&gt;<td&gt; 354236</td&gt;<td</td&gt;</tr&gt;</tbody&gt;</table

isolation of normal and cancer-associated fibroblasts from fresh tissues by fluorescence activated cell sorting (facs)

Associadas câncer fibroblastos (FAC), são o tipo de célula mais proeminente dentro do estroma tumoral de muitos cancros, em particular, carcinoma da mama, e a sua presença é muitas vezes associada proeminente com 1,2 prognóstico pobre. FAC são uma subpopulação de fibroblastos estromais activadas, muitos dos quais expressam o marcador de miofibroblastos α SMA-3. FAC originam a partir de fibroblastos dos tecidos locais, bem como a partir de medula óssea derivadas de células recrutadas para o tumor em desenvolvimento e adoptar um fenótipo CAF, sob a influência do microambiente do tumor 4. FAC foram mostrados para facilitar a iniciação do tumor, crescimento e a progressão através de sinalização que promove a proliferação de células de tumor, angiogénese e invasão 5-8. Nós demonstramos que a FAC aumentar o crescimento do tumor através da mediação de tumor promover a inflamação, a partir das primeiras fases de pré-neoplásicas 9. Apesar das evidências cada vez maior das FAC papel crucial na facilitação do crescimento tumoral,FAC estudam tem sido um desafio em curso, devido à falta de marcadores específicos do CAF ea heterogeneidade vasta destas células, com muitos subtipos co-existentes no microambiente do tumor 10. Além disso, estudos de fibroblastos in vitro é dificultado pelo facto de o seu perfil de expressão do gene é muitas vezes alterados em cultura de tecido 11,12. Para resolver este problema e permitir que o perfil de expressão gênica de imparcial fibroblastos de rato fresco e tecidos humanos, foi desenvolvido um método baseado em protocolos anteriores de fluorescência Ativado separação celular (FACS) 13,14. A nossa abordagem baseia-se na utilização PDGFRα como um marcador de superfície para isolar fibroblastos de rato fresca e tecidos humanos. PDGFRα é abundantemente expresso por ambos os fibroblastos normais e cafs 9,15. Este método permite o isolamento de populações puras de fibroblastos normais e cafés, incluindo, mas não limitado a α-SMA + activado miofibroblastos. Fibroblastos isolados podem então serutilizados para a caracterização e comparação da evolução da expressão do gene que ocorre no FAC durante a tumorigénese. Na verdade, nós e outros relataram perfil de expressão de fibroblastos isolados pela classificação celular 16. Este protocolo foi realizado com sucesso em isolar e perfil populações altamente enriquecido de fibroblastos dos tecidos da pele, pâncreas, mama e pulmão. Além disso, o nosso método também permite que a cultura de células seleccionadas, a fim de realizar experiências funcionais e para evitar a contaminação por células tumorais, o que é muitas vezes um grande obstáculo ao tentar FAC cultura.

Usando PDGFRα como um marcador para fibroblastos resulta em isolamento de populações altamente enriquecidas de fibroblastos de tecido. O nível de pureza após separação foi de 99%, tal como quantificado por tipo pós-análise (Figura 2A). Calculando a percentagem de contaminação de fibroblastos células não pela expressão relativa de genes específicos de células de controlo (Figura 2B) mostra tipicamente contaminação 0,1-0,6%. Este nível de pureza permite profiling transc...

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Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS

Os fibroblastos de câncer associados (FAC) facilitar tumor iniciação, crescimento e progressão através de sinalização que promove a proliferação, angiogênese e inflamação. Descrevemos aqui um método para isolar populações puras de fibroblastos normais e cafés de rato fresca e tecidos humanos por selecção de células, utilizando PDGFRα como um marcador de superfície.

Isolamento de fibroblastos normais e associados ao câncer de tecidos frescos, classificando células activadas por fluorescência (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their ...

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Biology

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

35.5K visualizações.  Tel Aviv University. Os fibroblastos de câncer associados (FAC) facilitar tumor iniciação, crescimento e progressão através de sinalização que promove a proliferação, angiogênese e inflamação. Descrevemos aqui um método para isolar populações puras de fibroblastos normais e cafés de rato fresca e tecidos humanos por selecção de células, utilizando PDGFRα como um marcador de superfície.

Vídeo: Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

Isolamento e purificação de Drosophila Neurônios periféricos por classificação Bead Magnética

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

Biologia

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

Separação de células de fluorescência Activated de Protoplastos Usina

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

Bromodeoxyuridine (BrdU) Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

Microbes are major agents mediating the degradation of numerous dissolved organic carbon (DOC) substrates in aquatic environments. However, identification of bacterial taxa that transform specific pools of DOC in nature poses a technical challenge. 
Here we describe an approach that couples bromodeoxyuridine (BrdU) incorporation, fluorescence activated cell sorting (FACS), and 16S rRNA gene-based molecular analysis that allows culture-independent identification of bacterioplankton capable of degrading a specific DOC compound in aquatic environments. Triplicate bacterioplankton microcosms are set up to receive both BrdU and a model DOC compound (DOC amendments), or only BrdU (no-addition control). BrdU substitutes the positions of thymidine in newly synthesized bacterial DNA and BrdU-labeled DNA can be readily immunodetected 1,2. Through a 24-hr incubation, bacterioplankton that are able to use the added DOC compound are expected to be selectively activated, and therefore have higher levels of BrdU incorporation (HI cells) than non-responsive cells in the DOC amendments and cells in no-addition controls (low BrdU incorporation cells, LI cells). After fluorescence immunodetection, HI cells are distinguished and physically separated from the LI cells by fluorescence activated cell sorting (FACS) 3. Sorted DOC-responsive cells (HI cells) are extracted for DNA and taxonomically identified through subsequent 16S rRNA gene-based analyses including PCR, clone library construction and sequencing.

Bromodeoxyuridine BrdU Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

Environmental bacterioplankton are incubated with a model dissolved organic carbon (DOC) compound and a DNA labeling reagent, bromodeoxyuridine (BrdU). Afterward, DOC-degrading cells are separated from the bulk community based on their elevated BrdU incorporation using fluorescence activated cell sorting (FACS). These cells are then identified by subsequent molecular analyses.

Bromodeoxiuridina Labeling (BrdU) e separação de células subseqüentes Fluorescence Activated de Cultura independente de Identificação de carbono orgânico dissolvido de degradação bacterioplâncton

Microbes are major agents mediating the degradation of numerous dissolved organic carbon (DOC) substrates in aquatic environments. However, identification ...

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

Liver fibrosis is defined by the excessive deposition of extracellular matrix by activated myofibroblasts. There are multiple precursors of hepatic myofibroblasts, including hepatic stellate cells, portal fibroblasts and bone marrow derived fibroblasts 1. Hepatic stellate cells have been the best studied, but portal fibroblasts are increasingly recognized as important contributors to the myofibroblast pool, particularly in biliary fibrosis 2. Portal fibroblasts undergo proliferation in response to biliary epithelial injury, potentially playing a key role in the early stages of biliary scarring 3-5. A method of isolating portal fibroblasts would allow in vitro study of this cell population and lead to greater understanding of the role portal fibroblasts play in biliary fibrosis.
Portal fibroblasts have been isolated using various techniques including outgrowth 6, 7 and liver perfusion with enzymatic digestion followed by size selection 8. The advantage of the digestion and size selection technique compared to the outgrowth technique is that cells can be studied without the necessity of passage in culture. Here, we describe a modified version of the original technique described by Kruglov and Dranoff 8 for isolation of portal fibroblasts from rat liver that results in a relatively pure population of primary cells.

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

A technique for isolating portal fibroblasts from rat liver is described. Livers are perfused and digested in situ with collagenase, followed by ex vivo digestion of the liver slurry and size selection of cells. This method provides a pure population of portal fibroblasts without the need for passage in culture.

Isolamento de fibroblastos de rato por Portal<em&gt; In situ</em&gt; Perfusão de fígado

Liver fibrosis is defined  by the excessive deposition of extracellular matrix by activated  myofibroblasts. There are multiple precursors of hepatic ...

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

Análise de célula específico<em&gt; Arabidopsis</em&gt; Folhas utilizando ordenação células activadas por fluorescência

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

Análise de suspensões celulares isoladas de tecidos sólidos por citometria de fluxo espectral

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

Magnético-ativado da pilha classificação estratégias para isolar e purificar sinovial fluidos derivados mesenquimais células-tronco de um modelo de coelho

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

Single Cell Micro-aspiration as an Alternative Strategy to Fluorescence-activated Cell Sorting for Giant Virus Mixture Separation

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or fluorescence activated cell sorting (FACS) applied to the viral population. However, when the viruses in the mixture have similar morphologic properties and one of the viruses multiplies slowly, the presence of two viruses is discovered at the stage of genome assembly and the viruses cannot be separated for further characterization. To solve this problem, we developed a single cell micro-aspiration procedure that allows for separation and cloning of highly similar viruses. In the present work, we present how this alternative strategy allowed us to separate the small viral subpopulations of Clandestinovirus ST1 and Usurpativirus LCD7, giant viruses that grow slowly and do not lead to amoebal lysis compared to the lytic and fast-growing Faustovirus. Purity control was assessed by specific gene amplification and viruses were produced for further characterization.

Here, we describe a single cell micro-aspiration method for the separation of infected amoebae. In order to separate viral subpopulations in Vermamoeba vermiformis infected by Faustoviruses and unknown giant viruses, we developed the protocol detailed below and demonstrated its ability to separate two low-abundance novel giant viruses.

Micro-aspiração de célula única como uma estratégia alternativa à classificação celular ativada por fluorescência para separação de mistura de vírus gigante

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or ...

Fluorescence-Activated Cell Sorting for the Isolation of Scleractinian Cell Populations

Coral reefs are under threat due to anthropogenic stressors. The biological response of coral to these stressors may occur at a cellular level, but the mechanisms are not well understood. To investigate coral response to stressors, we need tools for analyzing cellular responses. In particular, we need tools that facilitate the application of functional assays to better understand how cell populations are reacting to stress. In the current study, we use fluorescence-activated cell sorting (FACS) to isolate and separate different cell populations in stony corals. This protocol includes: (1) the separation of coral tissues from the skeleton, (2) creation of a single cell suspension, (3) labeling the coral cells using various markers for flow cytometry, and (4) gating and cell sorting strategies. This method will enable researchers to work on corals at the cellular level for analysis, functional assays, and gene expression studies of different cell populations.

Corals create biodiverse ecosystems important for both humans and marine organisms. However, we still do not understand the full potential and function of many coral cells. Here, we present a protocol developed for the isolation, labeling, and separation of stony coral cell populations.

Classificação celular ativada pela fluorescência para o isolamento de populações celulares escleracianas

Coral reefs are under threat due to anthropogenic stressors. The biological response of coral to these stressors may occur at a cellular level, but the ...

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 ...