Name: detection and isolation of apoptotic bodies to high purity
Uploaded: 2018-08-12T15:00:00.000+00:00
Duration: 12 min 17 s
Description: Watch this Scientific Journal Video about Detection and Isolation of Apoptotic Bodies to High Purity at JoVE.com

Isto trabalhou era suportada por concessões do nacional de saúde e Conselho de pesquisa médica (GNT1125033 e GNT1140187) e Australian Research Council (DP170103790) para I.K.H.P.

1. indução da apoptose
<ol><li>Centrifugar a amostra de célula a 300 x g por 5 min e descartar o sobrenadante para remover os detritos de células pré-existentes. Nota: Quando utilizar células aderentes, células de sementes com antecedência e lave com 1 x solução de tampão fosfato (PBS) antes da indução de apoptose.</li>
	<li>Determinar o número do celular e coletar as células. Nota: Dependendo do pós-isolamento ensaio, recomendamos um número de celular inicial de pelo menos 1 x 107 células.</li>
	<li>Resuspenda em mídia completa (respectivo meio contendo soro fetal bezerro de 10% (vol/vol), penicilina 50 UI/mL, 50 µ g/mL de mistura de estreptomicina) para uma concentração final de 1 x 106 células/mL.</li>
	<li>Alíquota ~ 2 x 106 células por poço de um prato bem 6.</li>
	<li>Para induzir apoptose, retire a tampa da placa e irradiar as células em 150 mJ/cm2 , usando uma difusora de UV. Isso deve levar cerca de 30-60 s. Nota: Antes da irradiação, certifique-se que ~0.5 x 106 células são retidas para o controle de célula 'Untreated'. Nota: Apoptose pode também ser induzidas através de outros métodos, como anti-Fas ou soro fome6.</li>
	<li>Incube a 37° C, 5% de CO2 para 2-8 horas, dependendo da linhagem celular.</li>
	<li>Usando um microscópio de luz superior do banco, visualize as células para confirmar a presença de apoptotic morfologias, como blebbing, formação de apoptopodia e formação de ApoBD. Nota: a ampliação de X 40 é suficiente</li>
	<li>Usando uma pipeta P1000, pipetar e coletar amostras de apoptotic.</li>
	<li>Lavar a placa com PBS 1x e combinam com a amostra restante para garantir o rendimento máximo.</li>
	<li>Coletar ~1/10th 'Toda Apoptotic no exemplo' (WAS) de controle.</li>
	<li>Colete a amostra 'Tratada'.</li>
	<li>Centrifugue o WAS e o Untreated amostras a 3.000 x g, durante 6 min.</li>
	<li>Resuspenda em 1 mL de 1X PBS e reserve no gelo.</li>
	<li>Para isolamento de ApoBD, continue a qualquer passo 2 ou 3 com a amostra de apoptotic existente.</li>
</ol>2. ApoBD isolamento através de FACS
<ol><li>Centrifugar a amostra inteira a 3.000 x g, durante 6 min.</li>
	<li>Remova a maioria do líquido sobrenadante sem perturbar o sedimento.</li>
	<li>Resuspenda em uma solução de coloração contendo 1 mL de 1 tampão de ligação x A5, 75 µ l de A5-FITC e 2 µ l de TO-PRO-3 por 1 x 107 células.</li>
	<li>Incube a amostra no escuro à temperatura ambiente por 10 min.</li>
	<li>Adicionar 1 a 2 mL de tampão de ligação 1 x A5 e centrifugar a amostra a 3.000 x g, durante 6 min remover a mancha excesso. Nota: Para populações de células mistas ou amostras de tecido, executar um anticorpo que mancha passo usando uma combinação de marcadores do tipo específico de célula em 1 tampão de ligação x A5 e incubar no gelo por 20 min (ou conforme o protocolo do fabricante) antes de centrifugação a 3.000 x g, durante 6 min.</li>
	<li>Resuspenda o pellet de amostra em 3 mL de tampão de FACS (1X PBS, 1 tampão de ligação x A5, 10% FSC, 2 mM EDTA) por 1 x 107 células.</li>
	<li>Filtrar através de um filtro de célula 70 µm para um fundo redondo, tubo de polipropileno (citometria de fluxo) e manter as amostras no gelo e no escuro.</li>
	<li>Ligar a máquina de FACS e executar conjunto padrão usando um bocal de 100 µm, executar o atraso de entrega e garantir um fluxo estável.</li>
	<li>Carregar a amostra e definir a velocidade de aquisição de ~ 1000 eventos/s.</li>
	<li>Ajustar o FSC, SSC, APC (PRO-TO-3) e tensões FITC (A5) e eventos de posição dentro as parcelas de FACS para garantir as populações podem ser claramente separados.</li>
	<li>Registre eventos de 20.000.</li>
	<li>Defina uma estratégia associada como em parte 4.</li>
	<li>No layout tipo, adicione o portão ApoBD final como a população de classificação desejada.</li>
	<li>Começam a adquirir a amostra e realizar uma espécie de teste através da recolha de 5.000-10.000 ApoBDs em um novo tubo contendo tampão de FACS ~ 250 µ l.</li>
	<li>Executar um sistema volta a descarga e carga classificada ApoBDs.</li>
	<li>Adquirir e gravar teste-tipo ApoBDs.</li>
	<li>Verifique a pureza de ApoBD é ~ 99% comparando FSC (eixo y) vs A5 (eventos de eixo x). Nota: A5 coloração pode reduzir ligeiramente quando re-analisar amostras devido à laser branqueamento.</li>
	<li>Depois de alta pureza é alcançada, carregar a amostra original e continuar a classificação até o número desejado de ApoBDs tenha sido obtido. Nota: Se necessário, dupla classificação pode ser realizada para isolar simultaneamente ApoCells e ApoBDs. Nota: Ao classificar por um longo período de tempo, recomenda-se Incubar o tubo de coleta a 4 ° C.</li>
	<li>Quando a classificação for concluído, coletar uma pequena parcela do pós-tipo ApoBDs, pós-classificar ApoCells, Untreated e WAS para validar a apoptose e confirmar pós-tipo pureza. Nota: Embora o teste-tipo e pós-tipo pureza não devem diferir significativamente, isto é baseado em configurações de fluxo e estabilidade.</li>
</ol>3. ApoBD isolamento através de centrifugação diferencial
<ol><li>Centrifugar a amostra de apoptotic existente em 300 x g durante 10 minutos.</li>
	<li>Recolher o sobrenadante, deixando ~ 500 µ l para não perturbar o centrifugado e adicionar em um novo tubo cônico de 15 mL.</li>
	<li>Remova os restantes 500 µ l e ressuspender as células em 2 mL de 1X PBS (o que representa a fração de' ApoCell-enriquecido'.</li>
	<li>Centrifugue o sobrenadante coletado por 20 min a 3.000 g.</li>
	<li>Procurem uma pelota e remova cuidadosamente o sobrenadante (líquido sobrenadante pode conter pequenas vesículas extracelulares, incluindo microvesicles e exosomes).</li>
	<li>Resuspenda o pellet em 1 mL de 1X PBS (o que representa a fração de 'ApoBD-enriquecido')</li>
	<li>Colete 100 µ l de cada viável, WAS, enriquecido em ApoCell e ApoBD-enriquecido de amostras em um novo fundo redondo, tubo de poliestireno (citometria de fluxo).</li>
	<li>Adicione 100 µ l de mancha contendo tampão de ligação 2 x A5, 1: 100 A5-FITC e 1:1,000 TO-PRO-3</li>
	<li>Incube a temperatura ambiente por 10 min no escuro.</li>
	<li>Analise as amostras por citometria de fluxo, usando a estratégia associada como descrito acima para validar a sucesso indução da apoptose e pureza da fração enriquecida com ApoBD.</li>
</ol>4. fluxo Cytometry Gating estratégia
<ol><li>Plot TO-PRO-3 (eixo y) contra FSC (eixo x) para separar as células necróticas (TO-PRO-3alta) de todos os outros eventos não-permeabilizado (TO-PRO-3baixo/intermediário).</li>
	<li>Selecione todos os eventos não-permeabilizado e plotar SSC contra A5. Portão de duas populações, incluindo população 1 (P1), SSCintermediário/alto, A5baixo/intermediário células e população 2 (P2), todos os outros eventos.</li>
	<li>De P2, plot TO-PRO-3 contra o A5 e selecione eventos deintermediário/alto A5 para excluir todos os detritos de células.</li>
	<li>Selecione todos os eventos positivos do A5 e plotar FSC contra A5. Separe (FSCbaixa) ApoBDs ApoCells (FSCintermediário/alto). Nota: Quando a retenção de ApoBDs para ApoBD de isolamento através da abordagem baseada em FACS, recomendamos uma etapa final selecionando ApoBDs e comparando TO-PRO-3 para A5 e selecionando todos os eventos. Isso garante que o portal classificação final usa fluorescência em vez de parâmetros FSC/SSC.</li>
	<li>Para análise de células viáveis, selecione P1 e execute uma das duas estratégias associadas. Para análise de células viáveis geral, plotar FSC contra A5 e selecione todas as células deintermediário/alto FSC, portanto, removendo detritos de células restantes. Como alternativa, para uma análise aprofundada, células viáveis podem ser separadas da A5– ApoCells início plotando TO-PRO-3 contra o FSC. Selecionar TO-PRO-3baixa, as células viáveis deintermediário/alto do FSC e TO-PRO-3intermediário, FSCintermediário/alto A5– ApoCells cedo.</li>
</ol>

<ol>
	<li>Poon, I. K., Lucas, C. D., Rossi, A. G., Ravichandran, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptotic+cell+clearance:+basic+biology+and+therapeutic+potential.">Apoptotic cell clearance: basic biology and therapeutic potential.</a> Nature Reviews. Immunology. 14, 166-180 (2014).</li><li>Atkin-Smith, G. K., Poon, I. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disassembly+of+the+Dying:+Mechanisms+and+Functions.">Disassembly of the Dying: Mechanisms and Functions.</a> Trends in Cell Biology. 27, 151-162 (2017).</li><li>Tixeira, R., 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=Defining+the+morphologic+features+and+products+of+cell+disassembly+during+apoptosis.">Defining the morphologic features and products of cell disassembly during apoptosis.</a> Apoptosis: An International Journal on Programmed Cell Death. 22, 475-477 (2017).</li><li>Sebbagh, 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=Caspase-3-mediated+cleavage+of+ROCK+I+induces+MLC+phosphorylation+and+apoptotic+membrane+blebbing.">Caspase-3-mediated cleavage of ROCK I induces MLC phosphorylation and apoptotic membrane blebbing.</a> Nature Cell Biology. 3, 346-352 (2001).</li><li>Coleman, M. L., 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=Membrane+blebbing+during+apoptosis+results+from+caspase-mediated+activation+of+ROCK+I.">Membrane blebbing during apoptosis results from caspase-mediated activation of ROCK I.</a> Nature Cell Biology. 3, 339-345 (2001).</li><li>Atkin-Smith, G. K., 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=A+novel+mechanism+of+generating+extracellular+vesicles+during+apoptosis+via+a+beads-on-a-string+membrane+structure.">A novel mechanism of generating extracellular vesicles during apoptosis via a beads-on-a-string membrane structure.</a> Nature Communications. 6, 7439 (2015).</li><li>Poon, I. K., 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=Unexpected+link+between+an+antibiotic,+pannexin+channels+and+apoptosis.">Unexpected link between an antibiotic, pannexin channels and apoptosis.</a> Nature. 507, 329-334 (2014).</li><li>Moss, D. K., Betin, V. M., Malesinski, S. D., Lane, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+role+for+microtubules+in+apoptotic+chromatin+dynamics+and+cellular+fragmentation.">A novel role for microtubules in apoptotic chromatin dynamics and cellular fragmentation.</a> Journal of Cell Science. 119, 2362-2374 (2006).</li><li>Kerr, J. F., Wyllie, A. H., Currie, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis:+a+basic+biological+phenomenon+with+wide-ranging+implications+in+tissue+kinetics.">Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics.</a> British journal of cancer. 26, 239-257 (1972).</li><li>Witasp, 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=Bridge+over+troubled+water:+milk+fat+globule+epidermal+growth+factor+8+promotes+human+monocyte-derived+macrophage+clearance+of+non-blebbing+phosphatidylserine-positive+target+cells.">Bridge over troubled water: milk fat globule epidermal growth factor 8 promotes human monocyte-derived macrophage clearance of non-blebbing phosphatidylserine-positive target cells.</a> Cell Death and Differentiation. 14, 1063-1065 (2007).</li><li>Orlando, K. A., Stone, N. L., Pittman, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rho+kinase+regulates+fragmentation+and+phagocytosis+of+apoptotic+cells.">Rho kinase regulates fragmentation and phagocytosis of apoptotic cells.</a> Experimental Cell Research. 312, 5-15 (2006).</li><li>Holmgren, L., 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=Horizontal+transfer+of+DNA+by+the+uptake+of+apoptotic+bodies.">Horizontal transfer of DNA by the uptake of apoptotic bodies.</a> Blood. 93, 3956-3963 (1999).</li><li>Zernecke, 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=Delivery+of+microRNA-126+by+apoptotic+bodies+induces+CXCL12-dependent+vascular+protection.">Delivery of microRNA-126 by apoptotic bodies induces CXCL12-dependent vascular protection.</a> Science Signaling. 2, ra81 (2009).</li><li>Schiller, 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=Autoantigens+are+translocated+into+small+apoptotic+bodies+during+early+stages+of+apoptosis.">Autoantigens are translocated into small apoptotic bodies during early stages of apoptosis.</a> Cell Death and Differentiation. 15, 183-191 (2008).</li><li>Elamin, M. H., 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=Curcumin+inhibits+the+Sonic+Hedgehog+signaling+pathway+and+triggers+apoptosis+in+medulloblastoma+cells.">Curcumin inhibits the Sonic Hedgehog signaling pathway and triggers apoptosis in medulloblastoma cells.</a> Molecular Carcinogenesis. 49, 302-314 (2010).</li><li>Sagulenko, V., 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=AIM2+and+NLRP3+inflammasomes+activate+both+apoptotic+and+pyroptotic+death+pathways+via+ASC.">AIM2 and NLRP3 inflammasomes activate both apoptotic and pyroptotic death pathways via ASC.</a> Cell Death and Differentiation. 20, 1149-1160 (2013).</li><li>Crescitelli, R., 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=Distinct+RNA+profiles+in+subpopulations+of+extracellular+vesicles:+apoptotic+bodies,+microvesicles+and+exosomes.">Distinct RNA profiles in subpopulations of extracellular vesicles: apoptotic bodies, microvesicles and exosomes.</a> Journal of Extracellular Vesicles. 2, (2013).</li><li>Turiak, L., 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=Proteomic+characterization+of+thymocyte-derived+microvesicles+and+apoptotic+bodies+in+BALB/c+mice.">Proteomic characterization of thymocyte-derived microvesicles and apoptotic bodies in BALB/c mice.</a> Journal of Proteomics. 74, 2025-2033 (2011).</li><li>Vermes, I., Haanen, C., Steffens-Nakken, H., Reutelingsperger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+assay+for+apoptosis.+Flow+cytometric+detection+of+phosphatidylserine+expression+on+early+apoptotic+cells+using+fluorescein+labelled+Annexin+V.">A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V.</a> Journal of Immunological Methods. 184, 39-51 (1995).</li><li>Jiang, L., 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=Monitoring+the+progression+of+cell+death+and+the+disassembly+of+dying+cells+by+flow+cytometry.">Monitoring the progression of cell death and the disassembly of dying cells by flow cytometry.</a> Nature Protocols. 11, 655-663 (2016).</li><li>Berda-Haddad, Y., 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=Sterile+inflammation+of+endothelial+cell-derived+apoptotic+bodies+is+mediated+by+interleukin-1alpha.">Sterile inflammation of endothelial cell-derived apoptotic bodies is mediated by interleukin-1alpha.</a> Proceedings of the National Academy of Sciences of the United States of America. 108, 20684-20689 (2011).</li><li>Lleo, 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=Shotgun+proteomics:+identification+of+unique+protein+profiles+of+apoptotic+bodies+from+biliary+epithelial+cells.">Shotgun proteomics: identification of unique protein profiles of apoptotic bodies from biliary epithelial cells.</a> Hepatology. 60, 1314-1323 (2014).</li><li>Atkin-Smith, G. K., 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=Isolation+of+cell+type-specific+apoptotic+bodies+by+fluorescence-activated+cell+sorting.">Isolation of cell type-specific apoptotic bodies by fluorescence-activated cell sorting.</a> Scientific Reports. 7, 39846 (2017).</li><li>Jiang, L., 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=Determining+the+contents+and+cell+origins+of+apoptotic+bodies+by+flow+cytometry.">Determining the contents and cell origins of apoptotic bodies by flow cytometry.</a> Scientific Reports. 7, 14444 (2017).</li></ol>

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Desde sua descrição inicial na década de 1950, campo da apoptose tem avançado consideravelmente, tornando-se uma área de pesquisa proeminente. Apesar do amplo interesse e grandes esforços, certos aspectos da apoptose, em particular a formação de ApoBDs, não foi bem estudaram devido a falta de metodologias adequadas. Estes incluem nomeadamente a limitação no acompanhamento da progressão de apoptose e ApoBD formação simultaneamente utilizando a citometria de fluxo tradicional análise A5/PI e as impurezas de ...

Apoptose, uma forma bem estudada de morte celular programada, é necessário para manter a homeostase fisiológica e remover células potencialmente prejudiciais dentro do corpo humano1. Após a indução da apoptose, pilhas apoptotic (ApoCells) podem se submeter a uma série de alterações morfológicas e desmontar em pequenas vesículas de membrana-limite denominadas ApoBDs. Em geral, este processo é conhecido como desmontagem de célula apoptótica e pode ser dividido em 3 etapas distintas, com base na morfologia2,3. Passo 1 (membrana plasmática blebbing) é caracterizado pela formação de balão-como estruturas na superfície das células, conhecido como blebs4,5. Passo 2 (formação de protrusão de apoptotic) inclui a formação de saliências longa membrana tais como apoptopodia, apoptopodia-frisado e microtubule picos6,7,8. Por último, etapa 3 (formação ApoBD) inclui a fragmentação do apoptotic saliências e/ou ApoCells para gerar ApoBDs6,9. Achados anteriores sugeriram um papel de ApoBDs em ajudar o afastamento de célula apoptótica e mediando a comunicação intercelular. Por exemplo, propõe-se que a fragmentação de um ApoCell em ApoBDs pode gerar pequenos pedaços 'pequenos' que podem ser facilmente removidos, envolvendo os fagócitos2,10,11. Além disso, os ApoBDs podem abrigar uma série de biomoléculas, tais como DNA, RNA e proteínas, que podem ser traficadas para células circundantes para facilitar a célula-célula comunicação12,13,14. Para funcionalmente investigar esses processos, é vital para confirmar três parâmetros-chave incluindo (i) validação de indução de apoptose e formação de ApoBD, (ii) isolamento de ApoBDs e (iii) confirmação da pureza de ApoBD.
Anteriormente, um número de métodos, incluindo citometria de fluxo e microscopia eletrônica tem sido costumava estudar apoptose e ApoBDs15,16,17,18. No entanto, quantificação e deteção de ApoBD são muitas vezes difíceis ou negligenciado. Por exemplo, a apoptose de baseados em citometria de fluxo mais rotineiramente usados do ensaio emprega anexina V (A5, uma proteína que se liga a exteriorizados ' comer-me' sinal fosfatidilserina (PtdSer)) e ácido nucleico mancha propidium iodeto (PI)19. No entanto, usando esta combinação de mancha universal, análise pressupõe que existem apenas três tipos de subconjuntos de células (células viáveis, ApoCells e necróticas células) na amostra. Além disso, embora considerado como "padrão ouro" por muitos pesquisadores por apoptose, ensaios de citometria de fluxo e análise de dados subsequentes muitas vezes exclui ApoBDs através de um passo inicial associado selecionar eventos deintermediário-elevado do FSC/SSC. Portanto, temos recentemente desenvolvido um ensaio de citometria de fluxo romance usando A5 e TO-PRO-3,7,20canais de outra mancha de ácidos nucleico pode ser seletivamente absorvida por caspase 3/7-ativado pannexin 1 (PANX1). Como caspase 3-induzida PANX1 ativação precede PtdSer exposição a fase inicial da apoptose, TO-PRO-3 manchas diferencialmente apoptotic e células necróticas. Além disso, essa abordagem combinada com nosso romance gating estratégia inclui eventos todos adquiridos durante a análise de dados e como resultado, seis subconjuntos de célula/partícula são identificados, incluindo: (i) células viáveis (FSC/SSCintermediário/alto, A5baixa , TO-PRO-3baixa), (ii) A5– ApoCells precoce (FSC/SSCintermediário/alto, A5baixo,intermediárioTO-PRO-3), (iii) A5+ ApoCells (FSC/SSCintermediário/alto, A5alta , TO-PRO-3intermediário), (iv) necróticas células ou tarde ApoCells (FSC/SSCintermediário/alto, A5alta,altaTO-PRO-3), (v) ApoBDs (FSC/SSCbaixa, A5intermediário, TO-PRO-3baixa / intermediárias) e (vi) detritos (FSC/SSCbaixa, A5baixo, TO-PRO-3baixo)20. Nossa abordagem enfatiza a importância de analisar todos os subconjuntos de células/partícula e, mais importante, a separação do ApoBDs de células e detritos20. Assim, essa abordagem demonstra uma técnica eficiente para validar a indução de apoptose e formação ApoBD simultaneamente.
Tradicionalmente, ApoBDs foram isolados através de uma variedade de abordagens de centrifugação diferencial, pelo qual ApoBDs podem ser separadas das células ou outras vesículas extracelulares, com base na densidade. No entanto, tais métodos de centrifugação são muitas vezes limitados pela baixa pureza de ApoBD, falta de uma etapa de quantificação para confirmar a pureza da amostra, e/ou incapacidade de separar as células específicas do tipo ApoBDs17,21,22. Portanto, desenvolvemos recentemente duas abordagens, uma baseada em FACS e uma nova diferencial centrifugação abordagem baseada que pode ser acoplada com o nosso método de citometria de fluxo estabelecido anteriormente para validar a indução de apoptose e amostra de pureza23. ApoBD isolamento através de nossa abordagem baseada em FACS pode enriquecer ApoBDs para até 99% de pureza e pode ser acoplado com uma variedade de anticorpos de tipo específico de célula para isolar ApoBDs de populações de células mistas, amostras de tecidos e fluidos corporais23. Além disso, nossa abordagem de centrifugação diferencial revisada demonstra um método eficiente para isolar ApoBDs a > 90% de pureza23.
Neste trabalho, descrevemos em detalhes nosso procedimento experimental para validar a indução de apoptose e para detectar e quantificar a formação de ApoBD. Os fluxos de trabalho de isolamento ApoBD usando métodos baseados em centrifugação de FACS-baseado e diferenciais também são elaborados e comparados. Os dados representativos demonstram que a metodologia descrita fornece uma ferramenta eficaz de ponta para estudos futuros ApoBD.

<table><tbody><tr><td>Cells e.g. cultured human THP-1 monocytes (clone number: TIB-202)</td><td>ATCC</td><td>-</td><td></td></tr><tr><td>RPMI 1640 medium</td><td>Life Technologies</td><td>22400-089</td><td></td></tr><tr><td>Penicillin-streptomycin mixture</td><td>Life Technologies</td><td>15140122</td><td></td></tr><tr><td>FSC</td><td>Gibco</td><td>10099-141</td><td></td></tr><tr><td>1x PBS</td><td>-</td><td>-</td><td></td></tr><tr><td>Annexin V FITC</td><td>BD Bioscience</td><td>-</td><td></td></tr><tr><td>TO-PRO-3 iodide</td><td>Life Technologies</td><td>T3605</td><td>TO-PRO-3 may cause skin, eye and respiratory irration. Avoid direct contact.</td></tr><tr><td>10x Annexin V binding buffer</td><td>BD Bioscience</td><td>556454</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>1001710526</td><td></td></tr><tr><td>Centrifuge tube (15 mL)</td><td>Cellstar</td><td>188271</td><td></td></tr><tr><td>Microcentrifuge tube (1.5 mL)</td><td>Sarstedt</td><td>72.690.001</td><td></td></tr><tr><td>Tissue culture incubator (37 &amp;deg;C, 5% CO2)</td><td>-</td><td>-</td><td></td></tr><tr><td>Centrifuge</td><td>Beckman Coulter</td><td>392932</td><td></td></tr><tr><td>FACS ARIA III Flow cytometer, configured with two lasers for FITC and APC detection</td><td>BD Bioscience</td><td>-</td><td></td></tr><tr><td>FACS Canto II Flow cytometer, configured with two lasers for FITC and APC detection</td><td>BD Bioscience</td><td>-</td><td></td></tr><tr><td>FACS Diva 6.1.1 software</td><td>BD Bioscience</td><td>-</td><td></td></tr><tr><td>FlowJo 8.8.6 software</td><td>-</td><td>-</td><td></td></tr><tr><td>&amp;nbsp;UV Stratalinker 1800</td><td>Stratagene</td><td>-</td><td></td></tr></tbody></table>

detection and isolation of apoptotic bodies to high purity

Corpos apoptotic (ApoBDs), microvesicles e exosomes são os principais membros da família vesículas extracelulares, com ApoBDs sendo um dos maior tipo. Tem sido proposto que a ApoBDs pode ajudar apuramento da célula, bem como a comunicação intercelular através do tráfico de biomoléculas. Abordagens convencionais utilizadas para a identificação e isolamento de ApoBDs são muitas vezes limitadas pela falta de quantificação exata e pureza da amostra de baixo. Aqui, descrevemos um fluxo de trabalho para confirmar a indução da apoptose, validar a formação ApoBD e isolar ApoBDs de alta pureza. Nós também irá delinear e comparar fluorescência-ativado da pilha (FACS) de triagem e centrifugação diferencial com base em abordagens para isolar ApoBDs. Além disso, a pureza de ApoBDs isoladas será confirmada usando um estabelecer previamente a coloração de baseados em citometria de fluxo e o método analítico. Tomados em conjunto, utilizando a abordagem descrita, THP-1 monócito apoptose e apoptose celular desmontagem foi induzida e validada, e ApoBD, gerado a partir de monócitos THP-1 foram isolados de uma pureza de 97-99%.

Usando o procedimento descrito aqui, THP-1 monócito apoptose foi induzido e ApoBDs foram detectados e isolados através um FACS-baseado ou uma abordagem de centrifugação diferencial (Figura 1). Em primeiro lugar, apoptose foi induzida por irradiação UV e as amostras foram coletadas após 2-3 h de incubação, quando demonstrado de células apoptóticas morfologias, incluindo blebbing, formação de protrusão da membrana apoptotic e a geração de ApoBDs...

Watch this Scientific Journal Video about Detection and Isolation of Apoptotic Bodies to High Purity at JoVE.com

Detection and Isolation of Apoptotic Bodies to High Purity

Um fluxo de trabalho usando a centrifugação diferencial ou citometria de fluxo é desenvolvido para detectar, quantificar e isolar corpos apoptotic de uma amostra de apoptotic de alta pureza.

Detecção e isolamento de corpos Apoptotic de alta pureza

Apoptotic bodies (ApoBDs), microvesicles and exosomes are the key members of the extracellular vesicle family, with ApoBDs being one of the largest type. ...

detection-and-isolation-of-apoptotic-bodies-to-high-purity

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

10.5K visualizações.  La Trobe University. Um fluxo de trabalho usando a centrifugação diferencial ou citometria de fluxo é desenvolvido para detectar, quantificar e isolar corpos apoptotic de uma amostra de apoptotic de alta pureza.

Vídeo: Detection and Isolation of Apoptotic Bodies to High Purity

Flow Cytometric Detection of Newly-formed Breast Cancer Stem Cell-like Cells After Apoptosis Reversal

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon named apoptosis reversal leads to increased tumorigenicity in various cell models under different stimuli. Previous studies have been focused on tracking this process in vitro and in vivo; however, the isolation of real reversed cells has yet to be achieved, which limits our understanding on the consequences of apoptosis reversal. Here, we take advantage of a Caspase-3/7 Green Detection dye to label cells with activated caspases after apoptotic induction. Cells with positive signals are further sorted out by fluorescence-activated cell sorting (FACS) for recovery. Morphological examination under confocal microscopy helps confirm the apoptotic status before FACS. An increase in tumorigenicity can often be attributed to the elevation in the percentage of cancer stem cell (CSC)-like cells. Also, given the heterogeneity of breast cancer, identifying the origin of these CSC-like cells would be critical to cancer treatment. Thus, we prepare breast non-stem cancer cells before triggering apoptosis, isolating caspase-activated cells and performing the apoptosis reversal procedure. Flow cytometry analysis reveals that breast CSC-like cells re-appear in the reversed group, indicating breast CSC-like cells are transited from breast non-stem cancer cells during apoptosis reversal. In summary, this protocol includes the isolation of apoptotic breast cancer cells and detection of changes in CSC percentage in reversed cells by flow cytometry.

Here, we present a protocol to isolate apoptotic breast cancer cells by fluorescence-activated cell sorting and further detect the transition of breast non-stem cancer cells to breast cancer stem cell-like cells after apoptosis reversal by flow cytometry.

Fluxo Cytometric deteção da recém-formada mama câncer células-tronco células após a reversão de apoptose

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon ...

Pesquisa do câncer

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depolarization

The central nervous system can experience a number of stresses and neurological insults, which can have numerous adverse effects that ultimately lead to a reduction in neuronal population and function. Damaged axons can release excitatory molecules including potassium or glutamate into the extracellular matrix, which in turn, can produce further insult and injury to the supporting glial cells including astrocytes and oligodendrocytes 8, 16. If the insult persists, cells will undergo programmed cell death (apoptosis), which is regulated and activated by a number of well-established signal transduction cascades 14. Apoptosis and tissue necrosis can occur after traumatic brain injury, cerebral ischemia, and seizures. A classical example of apoptotic regulation is the family of cysteine-dependent aspartate-directed proteases, or caspases. Activated proteases including caspases have also been implicated in cell death in response to chronic neurodegenerative diseases including Alzheimer's, Huntington's, and Multiple Sclerosis 4, 14, 3, 11, 7.
In this protocol we describe the use of the NucView 488 caspase-3 substrate to measure the rate of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte (OLG) cell cultures 15, 5, following exposure to different extracellular stresses such as high concentrations of potassium or glutamate. The conditionally-immortalized N19-OLG cell line (representing the O2A progenitor) was obtained from Dr. Anthony Campagnoni (UCLA Semel Institute for Neuroscience) 15, 5, and has been previously used to study molecular mechanisms of myelin gene expression and signal transduction leading to OLG differentiation (e.g.6, 10). We have found this cell line to be robust with respect to transfection with exogenous myelin basic protein (MBP) constructs fused to either RFP or GFP (red or green fluorescent protein) 13, 12. Here, the N19-OLG cell cultures were treated with either 80 mM potassium chloride or 100 mM sodium glutamate to mimic axonal leakage into the extracellular matrix to induce apoptosis 9. We used a bi-functional caspase-3 substrate containing a DEVD (Asp-Glu-Val-Asp) caspase-3 recognition subunit and a DNA-binding dye 2. The substrate quickly enters the cytoplasm where it is cleaved by intracellular caspase-3. The dye, NucView 488 is released and enters the cell nucleus where it binds DNA and fluoresces green at 488 nm, signaling apoptosis. Use of the NucView 488 caspase-3 substrate allows for live-cell imaging in real-time 1, 10. In this video, we also describe the culturing and transfection of immortalized N19-OLG cells, as well as live-cell imaging techniques.

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depola

Live-cell imaging of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte cell cultures using the NucView 488 caspase-3 substrate. This technique is applicable for programmed cell death assays in real-time in a variety of cell types and tissues.

Monitoramento de Atividades de caspase-3 Clivadas e apoptose das células oligodendrogliais Imortalizado usando Live-célula Imaging and Cleaveable Fluorogênico-dye seguintes substratos de potássio induzida por despolarização da membrana

The central nervous system can experience a number of stresses and neurological insults, which can have numerous adverse effects that ultimately lead to a ...

Neurociência

Flow Cytometric Analysis of Apoptotic Biomarkers in Actinomycin D-Treated SiHa Cervical Cancer Cells

Apoptosis biomarkers were investigated in actinomycin D-treated SiHa cervical cancer cells using a benchtop flow cytometer. Early biomarkers (Annexin V and mitochondrial membrane potential) and late biomarkers (caspases 3 and 7, and DNA damage) of apoptosis were measured in experimental and control cultures. Cultures were incubated for 24 hours in a humidified incubator at 37 &#176;C with 5% CO2. The cells were then detached using trypsin and enumerated using a flow cytometric cell count assay. Cells were further analyzed for apoptosis using an Annexin V assay, a mitochondrial electrochemical transmembrane potential assay, a caspase 3/7 assay, and a DNA damage assay. This article provides an overview of apoptosis and traditional flow cytometry, and elaborates flow cytometric protocols for processing and analyzing SiHa cells. The results describe positive, negative, and sub-optimal experimental data. Also discussed are interpretation and caveats in performing flow cytometric analysis of apoptosis using this analytical platform. Flow cytometric analysis provides an accurate measurement of early and late biomarkers for apoptosis.

Apoptosis can be characterized by flow cytometric analysis of early and late apoptotic biomarkers. The cervical cancer cell line, SiHa, was analyzed for apoptosis biomarkers after treatment with Actinomycin D using a benchtop flow cytometer.

Análise por citometria de fluxo de biomarcadores apoptóticos em células de câncer cervical SiHa tratadas com Actinomicina D

Apoptosis biomarkers were investigated in actinomycin D-treated SiHa cervical cancer cells using a benchtop flow cytometer. Early biomarkers (Annexin V ...

Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and decreases waste in costs and reagents. Here we describe optimization of a multiplex assay to assess apoptosis following a palmitic acid (PA) challenge in an in vitro hypothalamic model, using both fluorescent and luminescent based assays to measure viable cell counts and caspase-3/7 activity in a 96-well microtiter plate format. Following PA challenge, viable cells were determined by a resazurin-based fluorescent assay. Caspase-3/7 activity was then determined using a luminogenic substrate, DEVD, and normalized to cell number. This multiplexing assay is a useful technique for determining change in caspase activity following an apoptotic stimulus, such as saturated fatty acid challenge. The saturated fatty acid PA can increase hypothalamic oxidative stress and apoptosis, indicating the potential importance of assays such as that described here in studying the relationship between saturated fatty acids and neuronal function.

Multiplex assays can provide beneficial information for basic cellular mechanisms&#160;and eliminate waste of reagents and unnecessary repetitive experiments. We describe here a multiplex caspase-3/7 activity assay, using fluorescent- and luminescent-based methods, to determine cell viability in an in vitro hypothalamic model following oxidative challenge with palmitic acid.

Utilização de uma caspase Multiplexing ensaio para determinar a apoptose em um modelo de célula hipotalâmica

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and ...

Measuring Caspase Activity Using a Fluorometric Assay or Flow Cytometry

The activation of cysteine proteases, known as caspases, remains an important process in multiple forms of cell death. Caspases are critical initiators and executioners of apoptosis, the most studied form of programmed cell death. Apoptosis occurs during developmental processes and is a necessary event in tissue homeostasis. Pyroptosis is another form of cell death that utilizes caspases and is a critical process in activating the immune system through the activation of the inflammasome, which results in the release of members of the interleukin-1 (IL-1) family. To assess caspase activity, target substrates can be assessed. However, sensitivity can be an issue when examining single cells or low-level activity. We demonstrate how a fluorogenic substrate can be used with a population-based assay or single-cell assay by flow cytometry. With proper controls, different amino acid sequences can be used to identify which caspases are active. Using these assays, the simultaneous loss of the inhibitors of apoptosis proteins upon tumor necrosis factor (TNF) stimulation has been identified, which primarily induces apoptosis in macrophages rather than other forms of cell death.

The present protocol describes two methods to measure caspase activity through a fluorogenic substrate using flow cytometry or a spectrofluorometer.

Medindo a atividade da caspase usando um ensaio fluorométrico ou citometria de fluxo

The activation of cysteine proteases, known as caspases, remains an important process in multiple forms of cell death. Caspases are critical initiators ...

Bioquímica

Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death

Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely used in conjunction with Annexin V to determine if cells are viable, apoptotic, or necrotic through differences in plasma membrane integrity and permeability1,2. The Annexin V/ PI protocol is a commonly used approach for studying apoptotic cells3. PI is used more often than other nuclear stains because it is economical, stable and a good indicator of cell viability, based on its capacity to exclude dye in living cells 4,5. The ability of PI to enter a cell is dependent upon the permeability of the membrane; PI does not stain live or early apoptotic cells due to the presence of an intact plasma membrane 1,2,6. In late apoptotic and necrotic cells, the integrity of the plasma and nuclear membranes decreases7,8, allowing PI to pass through the membranes, intercalate into nucleic acids, and display red fluorescence 1,2,9. Unfortunately, we find that conventional Annexin V/ PI protocols lead to a significant number of false positive events (up to 40%), which are associated with PI staining of RNA within the cytoplasmic compartment10. Primary cells and cell lines in a broad range of animal models are affected, with large cells (nuclear: cytoplasmic ratios &lt;0.5) showing the highest occurrence10. Herein, we demonstrate a modified Annexin V/ PI method that provides a significant improvement for assessment of cell death compared to conventional methods. This protocol takes advantage of changes in cellular permeability during cell fixing to promote entry of RNase A into cells following staining. Both the timing and concentration of RNase A have been optimized for removal of cytoplasmic RNA. The result is a significant improvement over conventional Annexin V/ PI protocols (&lt; 5% events with cytoplasmic PI staining).

An accurate method for the assessment of cell death is described. The protocol improves upon conventional Annexin V/ propidium iodide (PI) protocols, which display up to 40% false- positive events in cell lines and primary cells from a broad range of animal models.

Modificado anexina V / propídio Assay Apoptose Iodeto para avaliação rigorosa da morte celular

Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely ...

Biologia

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

Quantificação de Efferocytosis por microscopia de fluorescência de célula única

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

Imunologia e Infecção

Experimental Analysis of Apoptotic Thymocyte Engulfment by Macrophages

Cell apoptosis is a natural process and plays a critical role in embryonic development, homeostatic regulation, immune tolerance induction, and resolution of inflammation. Accumulation of apoptotic debris in the body may trigger chronic inflammatory responses that lead to systemic autoimmune diseases over time. Impaired apoptotic cell clearance has been implicated in a variety of autoimmune diseases. Apoptotic clearance is a complex process rarely detected under physiological conditions. It involves abundant surface receptors and signaling molecules. Studying the process of apoptotic cell clearance provides insightful molecular mechanisms and subsequent biological responses, which may lead to the development of new therapeutics. Here, we describe protocols for the induction of apoptotic thymocytes, the preparation of peritoneal macrophages, and the analysis of apoptotic cell clearance by flow cytometry and microscopy. All cells will undergo apoptosis at a certain stage, and many residential and circulating cells can uptake apoptotic debris. Therefore, the protocol described here can be used in many applications to characterize apoptotic cell binding and ingestion by many other cell types.

Here, we present a protocol to prepare apoptotic thymocytes and peritoneal macrophages and analyze the efficiency of efferocytosis and the specific inhibitor-mediated blocking of apoptotic thymocytes engulfment. This protocol has a broad application in cell-mediated clearance of other particles including artificial beads and bacteria.

Análise experimental do engulfment do Thymocyte de apoptotic por macrófagos

Cell apoptosis is a natural process and plays a critical role in embryonic development, homeostatic regulation, immune tolerance induction, and resolution ...

Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important checkpoints of apoptosis, including mitochondrial fragmentation, release of mitochondrial cytochrome c into the cytosol, activation of caspases, chromatin condensation, DNA damage, nuclear fragmentation, plasma membrane blebbing, cell shrinkage, cell surface exposure of phosphatidylserine, and formation of apoptotic bodies. Anastasis can occur when apoptotic stimuli are removed prior to death, thereby allowing dying cells to reverse apoptosis and potentially other death mechanisms. Therefore, anastasis appears to involve physiological healing processes that could also sustain damaged cells inappropriately. The functions and mechanisms of anastasis are still unclear, hampered in part by the limited tools for detecting past events after the recovery of apparently healthy cells. Strategies to detect anastasis will enable studies of the physiological mechanisms, the hazards of undead cells in disease pathology, and potential therapeutics to modulate anastasis. Here, we describe effective strategies using live cell microscopy and a mammalian caspase biosensor&#160;for identifying and tracking anastasis in mammalian cells.

The term anastasis refers to the phenomenon in which dying cells reverse a cell suicide process at a late stage, repair themselves, and ultimately survive. Here we demonstrate protocols for detecting and tracking cells that undergo anastasis.

Estratégias para Rastreamento Anastasis, uma célula de sobrevivência Fenômeno que reverte apoptose

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important ...

Identification of Intracellular Signaling Events Induced in Viable Cells by Interaction with Neighboring Cells Undergoing Apoptotic Cell Death

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent live cells. Vital activities, such as survival, proliferation, growth, and differentiation, are among the many cellular functions modulated by apoptotic cells. The ability to recognize and respond to apoptotic cells appears to be a universal feature of all cells, regardless of lineage or organ of origination. However, the diversity and complexity of the response to apoptotic cells mandates that great care be taken in dissecting the signaling events and pathways responsible for any particular outcome. In particular, one must distinguish among the multiple mechanisms by which apoptotic cells can influence intracellular signaling pathways within viable responder cells, including: receptor-mediated recognition of the apoptotic cell, release of soluble mediators by the apoptotic cell, and/or engagement of the phagocytic machinery. Here, we provide a protocol for identifying intracellular signaling events that are induced in viable responder cells following their exposure to apoptotic cells. A major advantage of the protocol lies in the attention it pays to dissection of the mechanism by which apoptotic cells modulate signaling events within responding cells. While the protocol is specific for a conditionally immortalized mouse kidney proximal tubular cell line (BU.MPT cells), it is easily adapted to cell lines that are non-epithelial in origin and/or derived from organs other than the kidney. The use of dead cells as a stimulus introduces several unique factors that can hinder the detection of intracellular signaling events. These problems, as well as strategies to minimize or circumvent them, are discussed within the protocol. Application of this protocol should aid our expanding knowledge of the broad influence that dead or dying cells exert on their live neighbors, both in health and in disease.

Here, we present a protocol for the determination of intracellular signaling events induced in viable cells by physical interaction with adjacent dead or dying cells. The protocol focuses on signaling events induced by receptor-mediated recognition of the dead cells, as opposed to their phagocytic uptake or release of soluble mediators.

Identificação de sinalização intracelular Eventos induzida em células viáveis ​​pela interação com as células vizinhas Submetidos a apoptose celular Morte

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent ...

Detecção e isolamento de corpos Apoptotic de alta pureza

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Quantificação de Efferocytosis por microscopia de fluorescência de célula única

Análise experimental do engulfment do Thymocyte de apoptotic por macrófagos

Estratégias para Rastreamento Anastasis, uma célula de sobrevivência Fenômeno que reverte apoptose

Identificação de sinalização intracelular Eventos induzida em células viáveis pela interação com as células vizinhas Submetidos a apoptose celular Morte

Detecção e isolamento de corpos Apoptotic de alta pureza

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