Este trabalho foi financiado em parte com o apoio do Instituto de Koch Grant P30-CA 14051 (núcleo) do Instituto Nacional de câncer, pelos institutos nacionais de Grant saúde (22-CA206157 e GM118066) e Curt mármore Cancer Research Fund de Angelika Amon. Angelika Amon também é um investigador do Howard Hughes Medical Institute e o Glenn Foundation para investigação biomédica. S. S. foi apoiado pelo americano italiano Cancer Foundation (AICF), por uma bolsa de pesquisa do câncer de acções Marie Curie e a associação italiana para pesquisa de câncer (AIRC) e por uma bolsa de pesquisa de câncer quinquenal do KI. E.M. foi apoiado por uma bolsa do Howard Hughes Medical Institute.

Condição de cultura
Linhagem de células de RPE-1 hTERT foi cultivada na modificado Eagle suplementado de Dulbecco (Tabela de materiais) com 10% de soro Fetal bovino, 2 mM L-glutamina e 100 U/ml penicilina/estreptomicina. As células foram incubadas a 37 ° C com 5% CO2 em um ambiente umidificado. O protocolo descrito a seguir foi desenvolvido usando pratos de 10 cm e todos os detalhes técnicos relatados referem-se os pratos. Se usando pratos diferentes, escalar para cima ou para baixo em conformidade.
1. sincronização de células RPE-1
<ol><li>Use o recém descongeladas células que não tenham mais de 15 passagens para todos os experimentos. No dia 1, descongelar e dispensar ~2.5 - 5.0 x 105 RPE-1 hTERT células em um prato de cultura de tecido de 10 cm. Adicione 8 mL de meio de cultura padrão e incubar durante uma noite. Determine o número de célula, usando um contador de célula padrão (por exemplo, um hemocytometer). Nota: A densidade celular refere-se às condições de crescimento das células RPE-1. Condições de crescimento podem variar entre linhas de célula diferente; Considere-se ajustando-los adequadamente.</li>
	<li>No dia 2, sincronize pilhas na beira de G1/S por aspiração média de crescimento regular e adicionando meio de 8 mL contendo timidina de 5 mM. Nota: Concentração de timidina e duração do tratamento podem variar através das linhas de célula diferente. É aconselhável realizar experiências piloto para determinar as melhores condições para a linha de célula (s) a ser testado.</li>
	<li>No dia 3, 24 horas após a adição de timidina médio (etapa 1.2), lançamento do bloco de timidina por meio de aspiração e lavagem células três vezes com PBS 1x. Adicionar 8 mL do meio de crescimento regular e retornar as placas para a incubadora.</li>
</ol>2. geração de Aneuploid células por interferência com a atividade da quinase mitótica Mps1
<ol><li>No dia 3, 6h após o lançamento do bloco de timidina (etapa 1.3; isto é cerca de 3-6h antes que as células entrem na mitose), Aspire o meio, lave uma vez com PBS 1x e substituir com meio contendo 500 nM reversine (inibidor Mps1). Nota: As células de RPE-1 digite mitose 9-12 h após timidina wash-out10,11. No entanto, a cinética do ciclo celular variam entre linhagens celulares diferentes. Portanto, é crucial determinar esses cinética em experiências piloto, realizando análise de ciclo celular por métodos estabelecidos (por exemplo, medindo-se análise de conteúdo através de FACS DNA). Além disso, a concentração ideal de trabalho do inibidor de Mps1 pode variar entre linhas celulares. Experiências anteriores, utilizando células de RPE1 tem sido bem sucedidas usando reversine em uma concentração de trabalho de 500 nM10,11,12. É aconselhável realizar experiências piloto para determinar a concentração ideal para a linha de celular a ser testado.</li>
	<li>No dia 4, 12 h após o tratamento de reversine (cerca de 6 h depois de células em mitose), Aspire reversine médio e lavar as células três vezes com PBS 1x. Adicione 8 mL de meio de crescimento regular à placa e retornar as células para a incubadora.</li>
</ol>3. remoção de células de ciclismo Aneuploid e enriquecimento da população ArCK
<ol><li>No dia 6, cerca de 72 h após células entram a primeira mitose (passo 2.2; isto é cerca de 66 h após reversine wash-out, que corresponde a aproximadamente 2-3 ciclos de célula), Aspire médio, lave uma vez com PBS 1x e substituir com meio contendo 300 nM nocodazole. Nota: Trabalho recente mostrou que as células de RPE-1 abrigando cariótipos aneuploid genomically instáveis e prendam sua proliferação sobre ciclos de 2-3 célula após a primeira mitose defeituoso10. No entanto, esta cinética pode variar entre linhagens celulares diferentes. Portanto, é crucial realizar experiências piloto para determinar o momento adequado para a remoção de células de ciclismo aneuploid.</li>
	<li>No dia 7, 12 h após o tratamento nocodazole, Aspire médio e adicionar 3 mL de 1X PBS para a placa. Sacudi as células mitóticas tocando o lado da placa. Gire o prato entre cada torneira para permitir até mesmo remoção das células mitóticas.</li>
	<li>Para assegurar a completa remoção das células mitóticas, repita o processo de agitar-se por várias rodadas (3 - 5 vezes recomendadas), aspiração e adicionar 3 mL de 1X PBS entre cada rodada.</li>
	<li>Após agitação, delicadamente Aspire qualquer líquido que adere a tampa ou a borda da placa. Brevemente, verificar a placa sob um microscópio com ampliação de 10x. Certifique-se que algumas células mitóticas são observadas. Se mais de cinco células mitóticas são vistas ao abrigo do objectivo X 10, repita mais uma rodada de agitar-se. Nota: Células mitóticas apareceriam arredondadas e podem ser parcialmente desconectadas após agitar-se. É crucial assegurar a completa remoção das células mitóticas antes de expor as células para a próxima rodada de tratamento nocodazole. A não observância pode levar à morte celular mitótica e/ou resultar na geração de células tetraploide.</li>
	<li>Após agitar-off final, lave as células uma vez com 5 mL de 1X PBS. Adicione 8 mL de meio contendo 330 nM nocodazole a placa e incubar durante 12 h.</li>
	<li>Repeti nocodazole tratamento agitar-ro (passos 3.1-3.5) cada 12h até sem células mitóticas são observadas após a incubação. Geralmente, recomenda-se 4-5 rodadas de tratamento/agitar-se. Nota: As informações fornecidas aqui referir-se as células de RPE-1. O número de rodadas de tratamento agitar-ro pode variar entre linhagens celulares diferentes. Para evitar exposição desnecessária e prolongada, a nocodazole é crucial determinar cuidadosamente, em uma experiência piloto, o número de voltas necessárias para remoção eficiente e completa de células de ciclismo aneuploid.</li>
	<li>Depois de 4-5 rodadas de nocodazole tratamento agitar-ro (passos 3.1-3,6), adicionar 10 mL de meio de crescimento regular e incubar. Permitir que as células descansar por 12-24 h antes do uso futuro de manipulação ou ensaio. Nota: Após agitar-off final, as células restantes na placa são ciclo celular preso e, como mostrado recentemente10, abrigam cariótipos complexos. Estas células são denominadas células ArCK (preso com cariótipo complexo). É aconselhável realizar ensaios em populações de ArCK dentro de uma semana de isolamento.</li>
	<li>Opcionalmente, para avaliar a percentagem de células presas com cariótipos complexos, recolher e contar as células de cada shake-off usando um contador de célula padrão.</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57215/57215fig1.jpg"> Figura 1: esquemático geral do método usado para gerar e isolar aneuploid células com cariótipo complexo (ArCK) e imagens representativas. (A) esquema de ArCK células protocolo de geração e isolamento. (B) imagens representativas de células de hTERT RPE-1 em cada fase do tratamento. O último painel (esboçado em vermelho) representa as células isoladas de ArCK. Escala da barra 100 µm. <a href="//cloudflare.jove.com/files/ftp_upload/57215/57215fig1large.jpg" target="_blank">clique aqui para ver uma versão maior desta figura.</a>
4. Caracterização da população de ArCK
<ol><li>Confirme o sucesso isolamento da população ArCK realizando ensaios os seguintes.<ol>
<li>Bundas beta-galactosidase coloração (o marcador de senescência) usando um comercialmente disponível kit (veja a tabela de materiais).</li>
		<li>Medir a secreção de citocinas pró-inflamatórias, tais como CCL2, empregando kits comercialmente disponíveis (ver tabela de materiais).</li>
		<li>Confirme o aumento dos níveis do ciclo celular inibidores p53, p21, p16 por Western Blot. Nota: Para controlar adequadamente estes ensaios, ArCK populações devem ser comparadas às células de ciclismo euploid e aneuploid. É a primeira passagem baixa, selvagem-tipo RPE-1 hTERT células crescendo exponencialmente. Este último pode ser obtido por indução mis segregação de cromossomos em RPE-1 hTERT e colheita de células 72 h após a primeira mitose aberrante. Para fazer isso, siga o protocolo descrito acima de passo 1.1 colheita de células bem antes do primeiro tratamento de nocodazole (passo 3.1).</li>
	</ol>
</li>
	<li>Realize o sequenciamento de célula única para avaliar o cariótipo das células presos. Nota: O sequenciamento de célula única é um método bem estabelecido para determinar alterações cromossômicas e sub cromossômicas em toda uma população celular na única célula nível13. Os procedimentos detalhados de como realizar o sequenciamento de célula única podem ser encontrados em outro lugar10.</li>
</ol>

<ol><li>Santaguida, S., Amon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Short-+and+long-term+effects+of+chromosome+mis-segregation+and+aneuploidy%5BTitle%5D)+AND+%22Nature+Reviews+Molecular+Cell+Biology%22%5BJournal%5D)">Short- and long-term effects of chromosome mis-segregation and aneuploidy.</a> Nature Reviews Molecular Cell Biology. 16 (8), 473-485 (2015).</li>
<li>Pfau, S. J., Amon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chromosomal+instability+and+aneuploidy+in+cancer%3A+from+yeast+to+man%5BTitle%5D)+AND+%22EMBO+reports%22%5BJournal%5D)">Chromosomal instability and aneuploidy in cancer: from yeast to man.</a> EMBO reports. 13 (6), 515-527 (2012).</li>
<li>Gordon, D. J., Resio, B., Pellman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Causes+and+consequences+of+aneuploidy+in+cancer%5BTitle%5D)+AND+%22Nature+Reviews+Genetics%22%5BJournal%5D)">Causes and consequences of aneuploidy in cancer.</a> Nature Reviews Genetics. 13 (3), 189-203 (2012).</li>
<li>Holland, A. J., Cleveland, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Boveri+revisited%3A+chromosomal+instability%2C+aneuploidy+and+tumorigenesis%5BTitle%5D)+AND+%22Nature+Reviews+Molecular+Cell+Biology%22%5BJournal%5D)">Boveri revisited: chromosomal instability, aneuploidy and tumorigenesis.</a> Nature Reviews Molecular Cell Biology. 10 (7), 478-487 (2009).</li>
<li>Meena, J. K., Cerutti, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Telomerase+abrogates+aneuploidy-induced+telomere+replication+stress%2C+senescence+and+cell+depletion%5BTitle%5D)+AND+%22The+EMBO+Journal%22%5BJournal%5D)">Telomerase abrogates aneuploidy-induced telomere replication stress, senescence and cell depletion.</a> The EMBO Journal. , 1-14 (2015).</li>
<li>Ohashi, A., Ohori, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aneuploidy+generates+proteotoxic+stress+and+DNA+damage+concurrently+with+p53-mediated+post-mitotic+apoptosis+in+SAC-impaired+cells%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Aneuploidy generates proteotoxic stress and DNA damage concurrently with p53-mediated post-mitotic apoptosis in SAC-impaired cells.</a> Nature Communications. 6, 7668(2015).</li>
<li>Passerini, V., Ozeri-Galai, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+presence+of+extra+chromosomes+leads+to+genomic+instability%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">The presence of extra chromosomes leads to genomic instability.</a> Nature Communications. 7, 10754(2016).</li>
<li>Sheltzer, J. M., Blank, H. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aneuploidy+drives+genomic+instability+in+yeast%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Aneuploidy drives genomic instability in yeast.</a> Science. 333 (6045), 1026-1030 (2011).</li>
<li>Zhu, J., Pavelka, N., Bradford, W. D., Rancati, G., Li, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Karyotypic+determinants+of+chromosome+instability+in+aneuploid+budding+yeast%5BTitle%5D)+AND+%22PLoS+Genetics%22%5BJournal%5D)">Karyotypic determinants of chromosome instability in aneuploid budding yeast.</a> PLoS Genetics. 8 (5), e1002719(2012).</li>
<li>Santaguida, S., Richardson, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chromosome+Mis-segregation+Generates+Cell-Cycle-Arrested+Cells+with+Complex+Karyotypes+that+Are+Eliminated+by+the+Immune+System%5BTitle%5D)+AND+%22Developmental+Cell%22%5BJournal%5D)">Chromosome Mis-segregation Generates Cell-Cycle-Arrested Cells with Complex Karyotypes that Are Eliminated by the Immune System.</a> Developmental Cell. 41 (6), 638.e5-651.e5 (2017).</li>
<li>Santaguida, S., Vasile, E., White, E., Amon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aneuploidy-induced+cellular+stresses+limit+autophagic+degradation%5BTitle%5D)+AND+%22Genes+%26+Development%22%5BJournal%5D)">Aneuploidy-induced cellular stresses limit autophagic degradation.</a> Genes & Development. 29 (19), 2010-2021 (2015).</li>
<li>Santaguida, S., Tighe, A., D'Alise, A. M., Taylor, S. S., Musacchio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dissecting+the+role+of+MPS1+in+chromosome+biorientation+and+the+spindle+checkpoint+through+the+small+molecule+inhibitor+reversine%5BTitle%5D)+AND+%22The+Journal+of+Cell+Biology%22%5BJournal%5D)">Dissecting the role of MPS1 in chromosome biorientation and the spindle checkpoint through the small molecule inhibitor reversine.</a> The Journal of Cell Biology. 190 (1), 73-87 (2010).</li>
<li>Gawad, C., Koh, W., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+genome+sequencing%3A+current+state+of+the+science%5BTitle%5D)+AND+%22Nature+Reviews+Genetics%22%5BJournal%5D)">Single-cell genome sequencing: current state of the science.</a> Nature Reviews Genetics. 17 (3), 175-188 (2016).</li>
<li>López-Soto, A., Gonzalez, S., López-Larrea, C., Kroemer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunosurveillance+of+Malignant+Cells+with+Complex+Karyotypes%5BTitle%5D)+AND+%22Trends+in+cell+biology%22%5BJournal%5D)">Immunosurveillance of Malignant Cells with Complex Karyotypes.</a> Trends in cell biology. , 1-4 (2017).</li>
<li>Watson, E. V., Elledge, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aneuploidy+Police+Detect+Chromosomal+Imbalance+Triggering+Immune+Crackdown%21%5BTitle%5D)+AND+%22Trends+in+genetics%3A+TIG%22%5BJournal%5D)">Aneuploidy Police Detect Chromosomal Imbalance Triggering Immune Crackdown!</a> Trends in genetics: TIG. , 1-3 (2017).</li>
</ol>

Os autores não têm nada para divulgar.

Este novo método para gerar e enriquecer para células presas com cariótipos complexos (ArCK) permite o estudo de células que possuem vários cromossomo ganhos ou perdas e deixam de dividir. A configuração do método foi projetada para facilitar o isolamento das células ArCK de forma rápida e confiável.
O passo mais crítico neste ensaio é controlar rigorosamente o cronograma de tratamento medicamentoso e, mais importante ainda, a remoção de células de ciclismo aneuploid. Para obter melhores resultados, o tempo do tratamento nocodazole e agitar-off é de particular importância para garantir que as células de ciclismo são removidas da placa enquanto eles ainda são arredondadas e mitótica, impedindo a possibilidade de morte celular mitótica ou derrapagem em G1, potencialmente, criando uma população tetraploide. Não se recomenda que o timing de agitar-se afasta-se mais de duas horas de incubação recomendada 12-h nocodazole.
Futuros estudos sobre ArCK células têm o potencial de facilitar uma compreensão mais profunda de como complexo cariótipos afetam a fisiologia celular. Em particular, um estudo recente demonstrou que as células do sistema imunológico são capazes de interagir com e gatilho imune apuramento de ArCK células10. O método descrito aqui fornece um excelente ponto de partida para a caracterização adicional das células ArCK, incluindo o esclarecimento dos mecanismos moleculares subjacentes a autorização imune em células sem transformação e o estudo da transformação oncogênica como pode contornar este mecanismo de vigilância, uma questão crucial no campo14,15.

Erros no processo de segregação do cromossomo levam a aneuploidia, uma condição caracterizada por um número de cromossomos que não é um múltiplo do haploide1,2,3,4. Cariótipos aneuploid stress de replicação de gatilho que gera ainda mais instabilidade genômica5,6,7,8,9,10, aumenta complexidade de cariótipo e, finalmente, leva a detenção do ciclo celular de uma subpopulação de células10. O objetivo do método apresentado aqui é gerar e separar tal uma subpopulação de células de ciclismo. Empregando-se técnicas de cultura de tecidos de baixo custo, este protocolo facilita o isolamento e caracterização de células aneuploid ciclo celular-preso com cariótipos complexos. Estas células são denominadas células ArCK (preso com cariótipo complexo) e os seus homólogos de ciclismo euploid como controles.
Este protocolo é o primeiro a ser criado para o efeito e permite o isolamento e um estudo mais aprofundado das linhas ArCK, incluindo, mas não limitado a, sua senescência induzida e o fenótipo secretor associada a senescência (SASP), seus pro-inflamatórios características e sua capacidade de envolver-se com células do sistema imunológico. O método aqui apresentado foi desenvolvido em células humanas sem transformação, imortalizadas, mas ainda não foi testado em linhas de câncer. Algumas células transformadas podem ser insensíveis à detenção do ciclo celular devido à supressão de um ou vários caminhos; Portanto, ainda mais validação deve ser executada em outras linhas de célula.

<table><tbody><tr><td>DMEM (Dulbecco&amp;rsquo;s Modified Eagle Medium)&amp;nbsp;</td><td>Thermofisher</td><td>11995-073</td><td></td></tr><tr><td>Thymidine</td><td>Sigma Aldrich</td><td>T1859</td><td></td></tr><tr><td>Reversine</td><td>Cayman Chemical Company</td><td>10004412</td><td></td></tr><tr><td>Nocodazole</td><td>Sigma Aldrich</td><td>M1410</td><td></td></tr><tr><td>Anti-p53 antibody</td><td>Santa Cruz</td><td>sc-126</td><td></td></tr><tr><td>Anti-p21 antibody</td><td>Santa Cruz</td><td>sc-6246</td><td></td></tr><tr><td>Anti-p16 antibody</td><td>BD</td><td>554079</td><td></td></tr><tr><td>Senescence b-Galactosidase Staining Kit</td><td>Cell Signaling Technology&amp;nbsp;</td><td>9860</td><td></td></tr><tr><td>Proteome Profiler Human Cytokine Array Kit&amp;nbsp;</td><td>R&amp;amp;D Systems</td><td>ARY005B</td><td></td></tr></tbody></table>

generation and isolation of cell cycle-arrested cells with complex karyotypes

Segregação de mis cromossomo leva à aneuploidia, uma condição na qual células abrigam um número de cromossomas desequilibrado. Várias linhas de evidência indicam fortemente que aneuploidia provoca instabilidade do genoma, em última análise, gerando células com cariótipos complexos que prendam sua proliferação. Isolamento e caracterização de células abrigando cariótipos complexos são cruciais para estudar o impacto de um número de cromossomas desequilibrado na fisiologia celular. Até à data, não há métodos foram criados para isolar confiantemente tais células aneuploid. Este documento fornece um protocolo para o enriquecimento e a análise de células aneuploid com cariótipos complexos utilizando técnicas de cultura de tecidos de padrão, barato. Este protocolo pode ser usado para analisar várias características de células aneuploid com cariótipos complexos, incluindo seu fenótipo de secretora associada a senescência induzido, Propriedades pró-inflamatórias e capacidade de interagir com células do sistema imunológico. Porque as células cancerosas muitas vezes abrigam desequilíbrios no número de cromossomas, é crucial decifrar como aneuploidia impacta fisiologia celular em células normais, com o objetivo de desvendar os seus pro e anti tumorigenic efeitos.

Este método utiliza um sistema de cultura de tecidos em vitro para isolar células aneuploid com cariótipos complexos que prendam sua proliferação. Esta população é referida como células ArCK (preso com cariótipos complexos). A figura 1A mostra o esquema do experimento. Selvagem-tipo ciclismo RPE-1 hTERT células são sincronizadas na beira de G1/S com timidina e tratadas com um inibidor de Mps1 para induzir a segregação mis cromossomo. O eixo nocodazole veneno é adicionado às células 72 h após a indução da segregação mis cromossomo. 12 h após o tratamento de nocodazole, as células aneuploid que ainda são capazes de proliferar entre mitose e estão presos neste estágio do ciclo celular devido à interferência com a polimerização do microtúbulo. Estas preso células são então facilmente removidas por agitação suave. O processo de agitação é repetido 3 - 5 vezes para assegurar a remoção completa de células de ciclismo. A figura 1B mostra as imagens representativas de células RPE-1 em cada fase do tratamento.
ArCK células exibem aumento dos níveis de inibidores do ciclo celular, como a p21, p53 e p16 em comparação com euploid e aneuploid ciclismo células, como mostrado na Figura 2A. Além disso, a Figura 2B mostra β-galactosidase positivo mancha nas células ArCK em comparação com células de controle euploid, um marcador bem estabelecido para a senescência celular.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57215/57215fig2.jpg"> Figura 2: resultados representativos, utilizando células de ArCK. (A) Western blot avaliação níveis de inibidor do ciclo celular em células ArCK, aneuploid ciclismo e euploid. Os níveis de p53, p21, p16 e foram determinados por análise ocidental do borrão. Actina serviu como um controle de carregamento. (B) β-galactosidase, manchando a avaliação do nível de senescência em células ArCK. Atividade associada a senescência β-galactosidase (β-Gal) determinou-se em células euploid e células de ArCK. Escala da barra 100 µm. <a href="https://cloudflare.jove.com/files/ftp_upload/57215/57215fig2large.jpg" target="_blank">clique aqui para ver uma versão maior desta figura.</a>
Esta detenção do ciclo celular é uma característica proeminente de células aneuploid com cariótipos complexos, como demonstrado pela análise do cariótipo representativo de célula única sequenciamento na Figura 3, em que células de ArCK exibem Múltiplo, cromossomo aleatório de ganhos e perdas. Esta conclusão está em total acordo com anteriores relatórios10.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57215/57215fig3.jpg"> Figura 3: sequenciamento de célula única representante de células ArCK. Segmentação parcelas mostrando o cariótipo de seis células de ArCK representativos. Parcelas de segmentação mostram o número de cópia de todos os cromossomos de 1 a X em relação a uma referência euploid em uma escala de2 log (tipo selvagem RPE-1 é uma linhagem de células diploides de origem feminina). Ganhos do cromossomo são destacados em vermelho, perdas de cromossomo em verde. <a href="https://cloudflare.jove.com/files/ftp_upload/57215/57215fig3large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a>

Watch this Scientific Journal Video about Generation and Isolation of Cell Cycle-arrested Cells with Complex Karyotypes at JoVE.com

Generation and Isolation of Cell Cycle-arrested Cells with Complex Karyotypes

Aneuploidia leva à instabilidade do genoma, que eventualmente produz células de ciclo celular-preso com cariótipos complexos. Este artigo fornece um método simples e conveniente para isolar células aneuploid com cariótipos complexos que deixam de dividir.

Geração e isolamento de células de ciclo celular-preso com cariótipos complexos

Chromosome mis-segregation leads to aneuploidy, a condition in which cells harbor an imbalanced chromosome number. Several lines of evidence strongly ...

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Research

JoVE Journal

Cancer Research

10.4K visualizações.  Massachusetts Institute of Technology. Aneuploidia leva à instabilidade do genoma, que eventualmente produz células de ciclo celular-preso com cariótipos complexos. Este artigo fornece um método simples e conveniente para isolar células aneuploid com cariótipos complexos que deixam de dividir.

Vídeo: Generation and Isolation of Cell Cycle-arrested Cells with Complex Karyotypes

Studying Cell Cycle-regulated Gene Expression by Two Complementary Cell Synchronization Protocols

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such as cancer. Cell cycle-regulated gene expression analysis depends on cell synchronization into specific phases. Here we describe a method utilizing two complementary synchronization protocols that is commonly used for studying periodic variation of gene expression during the cell cycle. Both procedures are based on transiently blocking the cell cycle in one defined point. The synchronization protocol by hydroxyurea (HU) treatment leads to cellular arrest in late G1/early S phase, and release from HU-mediated arrest provides a cellular population uniformly progressing through S and G2/M. The synchronization protocol by thymidine and nocodazole (Thy-Noc) treatment blocks cells in early mitosis, and release from Thy-Noc mediated arrest provides a synchronized cellular population suitable for G1 phase and S phase-entry studies. Application of both procedures requires monitoring of the cell cycle distribution profiles, which is typically performed after propidium iodide (PI) staining of the cells and flow cytometry-mediated analysis of DNA content. We show that the combined use of two synchronization protocols is a robust approach to clearly determine the transcriptional profiles of genes that are differentially regulated in the cell cycle (i.e. E2F1 and E2F7), and consequently to have a better understanding of their role in cell cycle processes. Furthermore, we show that this approach is useful for the study of mechanisms underlying drug-based therapies (i.e. mitomycin C, an anticancer agent), because it allows to discriminate genes that are responsive to the genotoxic agent from those solely affected by cell cycle perturbations imposed by the agent.

We report two cell synchronization protocols that provide a context for studying events related to specific phases of the cell cycle. We show that this approach is useful for analyzing the regulation of specific genes in an unperturbed cell cycle or upon exposure to agents affecting the cell cycle.

Estudo da Expressão de Genes Reguladores do Ciclo Celular por dois Protocolos Complementares de Sincronização Celular

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such ...

Genética

Preparation of Primary Acute Lymphoblastic Leukemia Cells in Different Cell Cycle Phases by Centrifugal Elutriation

The ability to synchronize cells has been central to advancing our understanding of cell cycle regulation. Common techniques employed include serum deprivation; chemicals which arrest cells at different cell cycle phases; or the use of mitotic shake-off which exploits their reduced adherence. However, all of these have disadvantages. For example, serum starvation works well for normal cells but less well for tumor cells with compromised cell cycle checkpoints due to oncogene activation or tumor suppressor loss. Similarly, chemically-treated cell populations can harbor drug-induced damage and show stress-related alterations. A technique which circumvents these problems is counterflow centrifugal elutriation (CCE), where cells are subjected to two opposing forces, centrifugal force and fluid velocity, which results in the separation of cells on the basis of size and density. Since cells advancing through the cycle typically enlarge, CCE can be used to separate cells into different cell cycle phases. Here we apply this technique to primary acute lymphoblastic leukemia cells. Under optimal conditions, an essentially pure population of cells in G1 phase and a highly enriched population of cells in G2/M phases can be obtained in excellent yield. These cell populations are ideally suited for studying cell cycle-dependent mechanisms of action of anticancer drugs and for other applications. We also show how modifications to the standard procedure can result in suboptimal performance and discuss the limitations of the technique. The detailed methodology presented should facilitate application and exploration of the technique to other types of cells.

This protocol describes the use of centrifugal elutriation to separate primary acute lymphoblastic leukemia cells into different cell cycle phases.

Preparação de células de leucemia linfoblástica aguda primária em fases diferentes de ciclo celular por Elutriation centrífuga

The ability to synchronize cells has been central to advancing our understanding of cell cycle regulation. Common techniques employed include serum ...

Pesquisa do câncer

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral RNAs. When bound to viral double-stranded RNAs (dsRNAs), PKR undergoes dimerization and subsequent autophosphorylation. Phosphorylated PKR (pPKR) becomes active and induces phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2&#945;) to suppress global translation. Increasing evidence suggests that PKR can be activated under physiological conditions such as during the cell cycle or under various stress conditions without infection. However, our understanding of the RNA activators of PKR is limited due to the lack of a standardized experimental method to capture and analyze PKR-interacting dsRNAs. Here, we present an experimental protocol to specifically enrich and analyze PKR bound RNAs during the cell cycle using HeLa cells. We utilize the efficient crosslinking activity of formaldehyde to fix PKR-RNA complexes and isolate them via immunoprecipitation. PKR co-immunoprecipitated RNAs can then be further processed to generate a high-throughput sequencing library. One major class of PKR-interacting cellular dsRNAs is mitochondrial RNAs (mtRNAs), which can exist as intermolecular dsRNAs through complementary interaction between the heavy-strand and the light-strand RNAs. To study the strandedness of these duplex mtRNAs, we also present a protocol for strand-specific qRT-PCR. Our protocol is optimized for the analysis of PKR-bound RNAs, but it can be easily modified to study cellular dsRNAs or RNA-interactors of other dsRNA binding proteins.

We present experimental approaches for studying RNA-interactors of double-stranded RNA binding protein kinase RNA-activated (PKR) during the mammalian cell cycle using HeLa cells. This method utilizes formaldehyde to crosslink RNA-PKR complexes and immunoprecipitation to enrich PKR-bound RNAs. These RNAs can be further analyzed through high-throughput sequencing or qRT-PCR.

Estudando o RNA interactianos de proteína quinase RNA-ativado durante o ciclo celular dos mamíferos

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral ...

Bioquímica

Live Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

Imagem de célula viva de segregação do cromossomo durante a mitose

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

Biologia do Desenvolvimento

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture conditions they become prone to acquire chromosomal anomalies such as addition of whole or parts of chromosomes. Indeed, during long-term culture, karyotypic alterations are observed when enzymatic or chemical dissociation are used 2,3,4, while manual dissection of colonies for passaging retains a stable karyotype 5. Besides, changes in the environment such as the removal of feeder cells also seem to compromise the genetic integrity of hESC 3,6. Once chromosomal alterations could affect cellular physiology, the characterization of the genetic integrity of hESC in vitro is crucial considering hESC as an essential tool in embryogenesis studies and drug testing. Furthermore, for future therapeutic purposes chromosomal changes are a real concern as it is frequently associated to carcinogenesis.
Here we show a simple and useful method to obtain high quality chromosome spreads for subsequent analysis of chromosome set by G-banding, FISH, SKY or CGH techniques 7,8. We recommend checking the chromosomal status routinely with intervals of 5 passages in order to monitor the appearance of translocations and aneuploidies
Priscila Britto and Rafaela Sartore contributed equally to the paper.

Karyotyping is a simple and useful technique widely used for detecting genetic alterations. Here we describe a step by step protocol for chromosome spread preparation of human embryonic stem cells for monitoring the chromosomal status of these cells maintained in culture.

Preparação Spread cromossômica de células estaminais embrionárias humanas para Cariotipagem

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture ...

Biologia

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

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

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

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

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

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

Análise da posição do ciclo celular em células de mamíferos

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating cells. Cell cycle division is an integral part of growth and reproduction and deregulation of key cell cycle components have been implicated in the precipitating events of carcinogenesis 1,2. Molecular agents in anti-cancer therapies frequently target biological pathways responsible for the regulation and coordination of cell cycle division 3. Although cell cycle kinetics tend to vary according to cell type, the distribution of cells amongst the four stages of the cell cycle is rather consistent within a particular cell line due to the consistent pattern of mitogen and growth factor expression. Genotoxic events and other cellular stressors can result in a temporary block of cell cycle progression, resulting in arrest or a temporary pause in a particular cell cycle phase to allow for instigation of the appropriate response mechanism. 
The ability to experimentally observe the behavior of a cell population with reference to their cell cycle progression stage is an important advance in cell biology. Common procedures such as mitotic shake off, differential centrifugation or flow cytometry-based sorting are used to isolate cells at specific stages of the cell cycle 4-6. These fractionated, cell cycle phase-enriched populations are then subjected to experimental treatments. Yield, purity and viability of the separated fractions can often be compromised using these physical separation methods. As well, the time lapse between separation of the cell populations and the start of experimental treatment, whereby the fractionated cells can progress from the selected cell cycle stage, can pose significant challenges in the successful implementation and interpretation of these experiments.
Other approaches to study cell cycle stages include the use of chemicals to synchronize cells. Treatment of cells with chemical inhibitors of key metabolic processes for each cell cycle stage are useful in blocking the progression of the cell cycle to the next stage. For example, the ribonucleotide reductase inhibitor hydroxyurea halts cells at the G1/S juncture by limiting the supply of deoxynucleotides, the building blocks of DNA. Other notable chemicals include treatment with aphidicolin, a polymerase alpha inhibitor for G1 arrest, treatment with colchicine and nocodazole, both of which interfere with mitotic spindle formation to halt cells in M phase and finally, treatment with the DNA chain terminator 5-fluorodeoxyridine to initiate S phase arrest 7-9. Treatment with these chemicals is an effective means of synchronizing an entire population of cells at a particular phase. With removal of the chemical, cells rejoin the cell cycle in unison. Treatment of the test agent following release from the cell cycle blocking chemical ensures that the drug response elicited is from a uniform, cell cycle stage-specific population. However, since many of the chemical synchronizers are known genotoxic compounds, teasing apart the participation of various response pathways (to the synchronizers vs. the test agents) is challenging. 
Here we describe a metabolic labeling method for following a subpopulation of actively cycling cells through their progression from the DNA replication phase, through to the division and separation of their daughter cells. Coupled with flow cytometry quantification, this protocol enables for measurement of kinetic progression of the cell cycle in the absence of either mechanically- or chemically- induced cellular stresses commonly associated with other cell cycle synchronization methodologies 10. In the following sections we will discuss the methodology, as well as some of its applications in biomedical research.

Tracking subtle changes in the progression and kinetics of cell cycle stages can be accomplished by use of a combination of metabolic labeling of nucleic acids with BrdU and total genomic DNA staining via Propidium Iodide. This method avoids the need of chemical synchronization of cycling cells, thereby preventing the introduction of non-specific DNA damage, which in turn affects cell cycle progression.

Medindo a progressão do ciclo celular Cinética com marcação metabólica e Citometria de Fluxo

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating ...

Chromosome Preparation From Cultured Cells

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as fluorescence in situ hybridization (FISH), this assay has the powerful ability to analyze individual cells for aberrations that involve gains or losses of portions of the genome&#160;and rearrangements involving one or more chromosomes. In humans, chromosome abnormalities occur in approximately 1 per 160 live births1,2, 60-80% of all miscarriages3,4, 10% of stillbirths2,5, 13% of individuals with congenital heart disease6, 3-6% of infertility cases2, and in many patients with developmental delay&#160;and birth defects7. Cytogenetic analysis of malignancy is routinely used by researchers and clinicians, as observations of clonal chromosomal abnormalities have been shown to have both diagnostic and prognostic significance8,9.&#160; Chromosome isolation is invaluable for gene therapy and stem cell research of organisms including nonhuman primates and rodents10-13.
Chromosomes can be isolated from cells of live tissues, including blood lymphocytes, skin fibroblasts, amniocytes, placenta, bone marrow, and tumor specimens. Chromosomes are analyzed at the metaphase stage of mitosis, when they are most condensed and therefore more clearly visible. The first step of the chromosome isolation technique involves the disruption of the spindle fibers by incubation with Colcemid, to prevent the cells from proceeding to the subsequent anaphase stage. The cells are then treated with a hypotonic solution and preserved in their swollen state with Carnoy&#39;s fixative. The cells are then dropped on to slides and can then be utilized for a variety of procedures. G-banding involves trypsin treatment followed by staining with Giemsa to create characteristic light and dark bands. The same procedure to isolate chromosomes can be used for the preparation of cells for procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY)14,15.

Chromosomes can be isolated from live cells such as lymphocytes or skin fibroblasts, and from organisms including humans or mice. These chromosome preparations can be further utilized for routine G-banding and molecular cytogenetic procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY).

Cromossomo Preparação a partir de células cultivadas

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as ...

A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod shaped chromosomes which are moved individually by the mitotic spindle apparatus. Mitotic chromatin condensation yields chromosomes compacted fifty-fold denser as in interphase. During exit from mitosis, chromosomes have to re-establish their functional interphase state, which is enclosed by a nuclear envelope and is competent for replication and transcription. The decondensation process is morphologically well described, but in molecular terms poorly understood: We lack knowledge about the underlying molecular events and to a large extent the factors involved as well as their regulation. We describe here a cell-free system that faithfully recapitulates chromatin decondensation in vitro, based on mitotic chromatin clusters purified from synchronized HeLa cells and X. laevis egg extract. Our cell-free system provides an important tool for further molecular characterization of chromatin decondensation and its co-ordination with processes simultaneously occurring during mitotic exit such as nuclear envelope and pore complex re-assembly.

The molecular mechanisms of the decondensation of highly compacted mitotic chromatin are ill-defined. We present a cell-free assay based on mitotic chromatin clusters isolated from HeLa cells and Xenopus laevis egg extract that faithfully reconstitutes the decondensation process in vitro.

A Free Cell Ensaio para estudar Chromatin descondensação no fim da mitose

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod ...