The work was funded by the Leukemia and Lymphoma Society of the USA (6105-08), a Cancer Council NSW grant (13-02), an NHMRC Senior Research Fellowship (LJB) (1042305) and project grant (1041614).

O protocolo aqui descrito utiliza a linha celular de leucemia linfoblástica aguda NALM6 mas pode ser aplicada a outros tipos de células. 1. Soluções e Reagentes <ol><li> RPMI completo <ol><li> Adicionar 56 ml de soro fetal de vitelo (FCS) e 5,5 ml de 200 mM de L-glutamina a um frasco de 500 ml de meio RPMI-1640. </li></ol></li><li> BrdU da Solução <ol><li> Prepare 32,5 mM de BrdU (10 mg / ml) em fosfato de Dulbecco tamponado com solução salina (DPBS). </li></ol></li><li> BrdU completo RPMI <ol><li> Adicionar 6,2 ml de solução de estoque de BrdU para 10 ml de meio RPMI completo. </li></ol></li><li> Solução DNase <ol><li> Prepare 1 mg de ADNase / ml em DPBS. </li></ol></li><li> Tampão de Coloração <ol><li> Prepare a 3% de FCS inactivado pelo calor e azida de sódio 0,09% em DPBS. </li></ol></li><li> Consulte a Lista de Materiais para definições de Fixação Buffer, Permeabilização Tampão e tampão de lavagem. </li></ol> 2. Células NãoTe: As células não foram cultivadas durante mais de 6 meses. Este método é directamente adaptável a qualquer linha de células não-aderentes com os ajustes para a densidade de células e meios de cultura. Use de células que estão a crescer exponencialmente no início da experiência. <ol><li> Manter as células NALM6 em T-75 frascos de cultura em RPMI completo. Execute todas as etapas, em condições estéreis, utilizando uma classe II biossegurança gabinete. <ol><li> Manter células NALM6 entre 1-2 x 10 6 culas por ml, dividindo a cultura de três vezes por semana. </li><li> Incubar a 37 ° C em 5% de CO2 em ar. </li></ol></li></ol> 3. Pulso marcação das células com BrdU CUIDADO: Manusear com cuidado BrdU, pois é um agente mutagênico e teratogênico potencial. <ol><li> Centrifugar células a 150 xg durante 5 minutos. Nota: A transferência de células em meio fresco melhora a reprodutibilidade dos resultados. </li><li> Realizar uma contagem de células e as células ressuspender em completo RPMI a 2 x 10 6 cells / ml. </li><li> Dilui-se as células em 1 2 com BrdU RPMI completo produzindo uma concentração celular final de 1 x 10 6 células / ml. </li><li> Incubar a 37 ° C com 5% de CO 2 durante 45 min, depois dilui-se em 1 10 células com meio RPMI completo. Células centrifugar a 150 xg durante 5 min e cuidadosamente descartar todo o sobrenadante. </li><li> Ressuspender as células em um pequeno volume (~ 100 mL) de meio RPMI completo, executar uma contagem das células e ajusta-se 1 x 10 6 células / ml. </li><li> Pipetar 1 mL de células nos poços de uma placa de 48 poços. Pipeta de 1 ml de DPBS em todos os poços desocupadas para obter resultados mais reproduzíveis. </li><li> Incubar a 37 ° C em 5% de CO 2 no ar para timepoints desejados, aqui 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23 e 24 horas. Nota: O período de tempo vai depender do que o projeto experimental tem como objetivo medir. </li><li> Transferir todas as células em tubos de FACS utilizando uma pipeta. Lavar o bem sequencialmente com 1 ml volumes de PBS para um volume total final de 5 ml. </li><li> Centrifugar a 150 xg durante 5 min e cuidadosamente remover todo o sobrenadante. As células estão prontas para a coloração, executar esta (secção 4) imediatamente. </li></ol> 4. A coloração celular Nota: Se for necessária a coloração da superfície das células realizá-la antes da fixação, assegurando que as células são mantidas a 4 ° C durante toda. <ol><li> Ressuspender as células em 100 ul de tampão de coloração (por coloração da superfície opcional, adicionar o volume recomendado de anticorpo para antigénios de superfície e incuba-se durante 30 min a 4 ° C). </li><li> Adicionar 1 ml de tampão de coloração, centrifugar durante 5 minutos a 150 xg e desprezar o sobrenadante. Nota: anticorpo específico, a concentração, tempo de incubação, etc variará dependendo objectivos específicos experimentais. </li><li> Fixação e permeabilização <ol><li> Ressuspender as células em 100 ul de tampão de fixação e incubar durante 15 min à temperatura ambiente. </li><li> Adicionar 1 ml de of tampão de lavagem, centrifuga-se durante 5 min a 150 xg e desprezar o sobrenadante. </li><li> Ressuspender as células em 100 ul de tampão de permeabilização e incubar as células durante 10 minutos em gelo. </li><li> Adicionar 1 ml de tampão de lavagem, centrifugar durante 5 minutos a 150 xg, e desprezar o sobrenadante. </li><li> Ressuspender as células em 100 ul de tampão de fixação por tubo e incubar durante 5 min à temperatura ambiente. </li><li> Adicionar 1 ml de tampão de lavagem, centrifugar durante 5 minutos a 150 xg, e desprezar o sobrenadante. Nota: O protocolo pode ser pausado aqui se necessário. As células fixadas são estáveis ​​durante vários dias a 4 ° C, se ressuspensa em tampão de coloração. Remover o tampão de coloração após a centrifugação antes de prosseguir. </li></ol></li><li> O tratamento DNase <ol><li> Ressuspender as células em 100 ul de solução de ADNase (30 mg de ADNase / 10 6 células) e incubar as células durante 1 h a 37 ° C. </li><li> Adicionar 1 ml de tampão de lavagem, centrifugar a 150 xg durante 5 minutos e descartar o sobrenadante. </li&gt; </ol></li><li> Coloração de anticorpos Nota: A coloração para outros marcadores de BrdU intracelulares pode ser realizada em simultâneo com a coloração BrdU. <ol><li> IMPORTANTE: Prepare os controles de compensação consistem de células não coradas e as células marcadas com cada único fluorocromo. Idealmente, usar os mesmos anticorpos para os controlos de compensação, como os utilizados nos tubos experimentais. No entanto, se isso não for possível, os anticorpos para antigénios substitutos altamente expressos conjugado com o mesmo fluorocromo. </li><li> Ressuspender as células em 50 ul de tampão de lavagem e adicionar 1 mL / 10 6 células de anticorpo BrdU. Nota: pode também ser adicionado directamente conjugado anticorpos para outros antigénios intracelulares específicos. NOTA:. Os anticorpos para histona H3 fosforilada em Ser10 pode ser utilizado para discriminar entre células em G2 e M, histona H3 é fosforilada em Ser10 durante a mitose 10 Os anticorpos para cdc2 fosforilada em Tyr15 pode ser usado para detectar células que o VHAe compromete-se a mitose. 11 </li><li> Incubam-se as células durante 20 minutos à temperatura ambiente. </li><li> Adicionar 1 ml de tampão de lavagem, as células de centrifugação a 150 xg durante 5 minutos e descartar o sobrenadante. </li></ol></li></ol><ol start="6"><li> DNA Stain para análise do ciclo celular <ol><li> Soltar sedimento e adicionar 20 uL da solução de 7-AAD (0,25 ug). Nota: É crítico para utilizar uma quantidade constante de 7-AAD / célula. </li><li> Ressuspender as células em 1 ml de tampão de coloração. </li></ol></li></ol> 5. Recolha de Citometria de Fluxo de Dados Nota: A máquina necessária vai depender da quantidade e natureza dos fluorocromos utilizados. <ol><li> Recolher os seguintes parâmetros: o FSC-A, SSC-A, o FSC-H (FSC-W pode ser utilizado em vez do FSC-H) e 7-AAD fluorescência numa escala linear. Recolhe-se o canal de APC numa escala logarítmica. Colete quaisquer canais adicionais necessários para a avaliação dos rótulos superficiais ou internos utilizando uma escala logarítmica. </li><li> Execute comcompensação de sobreposição de sinais em espectros de emissão observada entre diferentes fluorocromos antes de analisar as amostras. Nota: A maioria dos citómetros de fluxo fará isso automaticamente. </li><li> Recolher pelo menos 10.000 eventos para cada amostra. </li></ol> 6. Análise de citometria de fluxo de dados Nota: FlowJo foi utilizado neste estudo de citometria de fluxo para a análise de dados, mas outros pacotes de software também pode ser usado. A estratégia de propagação é ilustrado na Figura 1. <ol><li> Identificar a população de células viáveis ​​utilizando parâmetros FSC-A e SSC-A. </li><li> Dentro desta população excluir dupletos e agregados utilizando FSC-A e FSC-H (FSC-W também podem ser usados ​​aqui). </li><li> Dentro desta população definir um gráfico de pontos utilizando 7-AAD no eixo dos x e BrdU-APC no eixo dos y. </li></ol><img alt="Figura 1" src="/files/ftp_upload/52840/52840fig1highres.jpg" width="700" /> Figura 1: Estratégia Gating Esquerda pa.nel: células ungated são mostrados em um FCS-A vs. SSC-A gráfico de pontos. A população de células viáveis ​​é identificado pela porta mostrado. Centro painel: células fechadas a partir do painel esquerdo são mostrados em uma FSC-A vs. FSC-H gráfico de pontos (FSC-W pode ser usado em vez de altura). Parelhas e agregados são identificados e excluídos pelo portão mostrado. Painel direito: células fechadas a partir da data de exclusão gibão no painel central são mostrados em um 7-AAD vs. APC-A gráfico de pontos. O anticorpo é marcado com BrdU APC permitindo a identificação de células que incorporaram BrdU durante a marcação por pulsos. 7-AAD fornece informações sobre o conteúdo de DNA. O portão superior define células positivas para BrdU e, portanto, na fase S no momento do pulso de BrdU, o portão inferior esquerdo, as células em G 0/1 ea porta inferior direito aqueles em G2 / M. <a href="https://www.jove.com/files/ftp_upload/52840/52840fig1highres.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <ol start="4"><li> Um ciclo celular<html> nálise <ol><li> Abra o ficheiro de dados e primeira porta nas células do portão exclusão dupleto. </li><li> Analisar essa população para distribuição do ciclo celular (localizado sob plataformas em software Flojo) e usar o modelo de Dean-Jett-Fox. </li><li> Obter as posições do G 0/1 e G 2 / M picos utilizando criar portões. </li><li> Porta nas células BrdU positivas e sujeita essa população a mesma análise do ciclo celular. </li><li> Forneça as posições para o G 0/1 e G 2 / M picos aplicando os mesmos portões de criar portas e definir restrições (usando as portas criadas) para as posições do G 0/1 e G 2 / M picos. Isto é ilustrado nos primeiros dois painéis da Figura 2. Nota: Outro software também pode ser usado para analisar os dados e as instruções iria variar em conformidade. </li></ol></li></ol>840 / 52840fig2highres.jpg &quot;width =&quot; 700 &quot;/&gt; Figura 2:. Progressão do Ciclo Celular O primeiro painel (todas as células) é fechado na população de células definida pela porta exclusão dupleto. Esta população foi exibida num histograma com 7-AAD no eixo dos X. O pico do pico G 0/1 é indicado pela seta por baixo do eixo. Em subsequentes painéis de células positivas para BrdU foram fechado em, como mostrado na Figura 1. O valor para a posição de 0/1 L obtido quando gating da porta exclusão dupleto é aplicada às células positivas para BrdU fechado dentro FlowJo software do ciclo celular. Cada painel posterior foi fechado sobre a população de BrdU positiva, como mostrado na Figura 1 e a posição do pico G 0/1 com base no valor obtido na análise de toda a população, como mostrado nos primeiros dois painéis. Utilizando a fracção negativa BrdU para identificar a localização da população L 0/1 para as células positivas para BrdU no mesmo SAMPle controla por quaisquer ligeiras diferenças na intensidade da mancha de ADN entre as amostras. O número mostrado em cada painel representa o tempo que o pulso de BrdU terminou. As fases do ciclo celular calculados são mostrados em verde com sombra. <a href="https://www.jove.com/files/ftp_upload/52840/52840fig2highres.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a>

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The authors have nothing to disclose.

A capacidade de analisar o ciclo celular é importante para a compreensão da biologia do cancro e o mecanismo de acção de ambas as drogas e os genes que influenciam a proliferação das células e o crescimento. Embora haja um grande número de ensaios que medem a proliferação celular supostamente, a maioria só fornecem uma medida que indica as células Número presente. Estes incluem ensaios que medem o número de células por visualização direta e contando, atividade metabólica ou concentração de ATP. A pri...

A avaliação das características do ciclo celular e alterações que ocorrem nas células durante a progressão do ciclo celular é fundamental para a compreensão de diversos aspectos da biologia, particularmente biologia do cancro. Muitos agentes em desenvolvimento para o tratamento de doenças malignas têm efeitos profundos sobre a progressão do ciclo celular ou induzir a morte de células através do ciclo celular dependente de mecanismos. A fim de estudar a dinâmica do ciclo celular ou células numa fase específica do ciclo celular, é usual para sincronizar as células. No entanto os métodos de sincronização pode ter efeitos prejudiciais sobre as células a ser estudada, potencialmente confundindo os resultados obtidos. 1 análise Recentemente, a utilização de proteínas marcadas com fluorescência que só estão presentes em fases específicas do ciclo de células têm permitido da progressão do ciclo celular em células individuais ao longo do tempo 2, no entanto as células a serem estudadas necessidade de ser geneticamente manipuladas para expressar estas proteínas marcadas, o que limita a sua utilização para sistemas em que este pode ser lidoily alcançado. O ciclo celular consiste de duas fases activas: a fase de síntese (s), onde o DNA é replicado e mitose (M), onde ocorre a divisão celular. Estas fases são separadas por três fases de hiato, 0 G, G 1 e G 2. G 0 ou quiescência, é uma fase de repouso, onde a célula tem deixado o ciclo, L é um onde as células aumentam de tamanho antes da replicação de ADN e L 2, onde o crescimento celular continua entre a conclusão da replicação de ADN, mas antes da divisão celular. A progressão através do ciclo celular é controlado por um número de pontos de verificação. O G 1 ponto de verificação é ativada quando as condições ambientais não são favoráveis ​​a síntese de DNA e impede a entrada na fase S. O intra-S fase checkpoint ou atraso pode ser desencadeada por danos no DNA que pode resultar em forquilhas de replicação paralisadas. Durante G 2 a fidelidade do DNA replicado é confirmada e se o dano for detectado, em seguida, o G2 </sub&gt; ponto de verificação é ativada permitindo o reparo do DNA antes da divisão celular. Um ponto de verificação final durante a mitose assegura que os cromatídeos foram correctamente alinhado com a placa mitótico modo que a divisão celular pode ser completada com êxito. 3 A activação destes pontos de verificação é normalmente usado para sincronizar as populações de células. Checkpoints do ciclo celular pode ser activada por um número de factores, mas na biologia do cancro a mais comum é a detecção de danos no DNA. A resposta a danos no ADN é iniciada pela PI3-quinase-quinases como ataxia e telangiectasia Rad3 relacionado (ATR) e a ataxia telangiectasia mutada (ATM) que activam as cinases efectoras a jusante Chk1 e Chk2, respectivamente. 3 Uma série de eventos activa Chk1 incluindo parado forquilhas de replicação, ligações cruzadas de DNA e danos da radiação ultravioleta enquanto Chk2 é ativado principalmente por quebras de cadeia dupla. O método usual para estudar o efeito de condições alteradas no comprimento do ciclo celular is para sincronizar as células numa fase específica do ciclo celular. 1 Isto pode ser alcançado através de vários métodos. As células podem ser fisicamente separadas com base no tamanho, densidade, dispersão lateral (granulosidade), e expressão de marcadores de superfície celular. Mais praticamente, as células podem ser sincronizado por meios químicos. Vários agentes, tais como timidina, hidroxiureia e arabinósido de citosina podem ser utilizados para inibir a síntese de ADN em fase S do ciclo celular, resultando numa acumulação de células em fase S que continuam ciclismo após os agentes são removidos. As células tratadas com nocodazole, o que impede a formação do fuso mitótico, parada com uma L 2 - ou M-fase teor de ADN. Eliminação do soro do meio de cultura resulta na acumulação de células na fase G 0. A re-adição de nutrientes dentro das séricos cultura re-inicia o ciclo normal das células. No entanto, todos esses métodos de sincronização interferir com ciclos normais e o crescimento de células e pode Result em morte celular significativa. A sincronização das células de leucemia linfoblástica aguda é particularmente difícil e estas células não são passíveis de manipulação genética. O método aqui descrito permite a avaliação da dinâmica do ciclo celular e o estudo de células em fases específicas do ciclo celular, sem sincronização tradicional ou modificação genética. Este método também pode ser útil para outros tipos de células em que a modificação genética e procedimentos de sincronização tradicionais não são prontamente obtidos. O método baseia-se no uso há muito estabelecida de bromodesoxiuridina (BrdU) a incorporação, o que tem muito pouco impacto sobre o crescimento a curto prazo e a proliferação de células. 4 protocolos BrdU Estabelecida tirar vantagem da incorporação de BrdU no ADN sintetizado de novo durante a fase S . Isto marca permanentemente células como estando em fase S durante a exposição BrdU. Esta população pode ser identificado em pontos de tempo mais tardios por coloração para BrdU Incorporção e, assim, agir como uma população sincronizados que podem ser seguidos e avaliados ao longo do tempo permitindo o estudo dos efeitos da droga sobre o trânsito do ciclo celular. BrdU precisa de ser exposto antes da coloração do anticorpo, geralmente atingidos após a ADNase ou tratamento ácido. 6,7 utilizando citometria de fluxo para detectar BrdU incorporada possibilita a inclusão de marcadores adicionais. O mais importante é a utilização de corantes para medir o teor de ADN, permitindo a avaliação da distribuição de fases do ciclo celular das células que estavam na fase S no início do estudo. 8 antigénios de superfície ou intracelulares adicionais Além disso, também pode ser estudada. 9 Estes pode referir-se os eventos do ciclo celular, tais como Ki67 ou características celulares para aparentemente não relacionadas, tais como marcadores de apoptose como a caspase-3 clivada. As aplicações potenciais são limitadas pela imaginação do investigador.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>APC BrdU Flow Kit</td>
			<td>BD Biosciences</td>
			<td>552598</td>
			<td>Contains BrdU antibody, 7-AAD and BD Cytofix/Cytoperm 
			Buffer (referred to as Fixation Buffer)</td>
		</tr>
		<tr>
			<td>BD Cytoperm Permeabilization Buffer Plus</td>
			<td>BD Biosciences</td>
			<td>561651</td>
			<td>Referred to as Permeabilization buffer</td>
		</tr>
		<tr>
			<td>BD Perm/Wash Buffer</td>
			<td>BD Biosciences</td>
			<td>554723</td>
			<td>Referred to as Wash buffer</td>
		</tr>
		<tr>
			<td>DNase</td>
			<td>Sigma</td>
			<td>D-4513</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Falcon 12 x 75-mm FACS tubes</td>
			<td>BD Biosciences</td>
			<td>352008</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Pharmingen Stain Buffer</td>
			<td>BD Biosciences</td>
			<td>554656</td>
			<td></td>
		</tr>
		<tr>
			<td>BD LSR FORTESSA flow cytometer</td>
			<td>BD Biosciences</td>
			<td>FORTESSA</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetman</td>
			<td>Gilson</td>
			<td>P2, P20, P100, P1000</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 w/o L-Gln 500 ml</td>
			<td>Lonza</td>
			<td>12-167F</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Lonza</td>
			<td>17-512F</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>FisherBiotec</td>
			<td>FBS-7100113</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Sigma</td>
			<td>G7513-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>5-Bromo-2&#8242;-deoxyuridine</td>
			<td>Sigma</td>
			<td>B5002-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon TC 150cm2 vented Flasks</td>
			<td>BD Biosciences</td>
			<td>355001</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes 25mL</td>
			<td>Greiner</td>
			<td>760180</td>
			<td></td>
		</tr>
		<tr>
			<td>Aersol Pipettes 200&#181;L</td>
			<td>Interpath</td>
			<td>24700</td>
			<td></td>
		</tr>
		<tr>
			<td>Aersol Pipettes 1mL</td>
			<td>Interpath</td>
			<td>24800</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Spintron</td>
			<td>GT-175R</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Binder</td>
			<td>C 150</td>
			<td></td>
		</tr>
		<tr>
			<td>AF488 anti-Histone H3 Phospho (Ser10) Antibody</td>
			<td>Cell Signalling</td>
			<td>9708S</td>
			<td></td>
		</tr>
		<tr>
			<td>Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb</td>
			<td>Cell Signalling</td>
			<td>2197S</td>
			<td></td>
		</tr>
		<tr>
			<td>Phospho-Chk1 (Ser345) (133D3) Rabbit mAb</td>
			<td>Cell Signalling</td>
			<td><a name="RANGE!C23">2348S</a></td>
			<td></td>
		</tr>
		<tr>
			<td>NALM6&#160;</td>
			<td>DSMZ</td>
			<td>ACC-128</td>
			<td></td>
		</tr>
	</tbody>
</table>

temporal tracking of cell cycle progression using flow cytometry without the need for synchronization

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell synchronization can be difficult for many cell systems. Standard practice is to block cell cycle progression at a specific stage and then release the accumulated cells producing a wave of cells progressing through the cycle in unison. However, some cell types find this block toxic resulting in abnormal cell cycling, or even mass death. Bromodeoxyuridine (BrdU) uptake can be used to track the cell cycle stage of individual cells. Cells incorporate this synthetic thymidine analog, while synthesizing new DNA during S phase. By providing BrdU for a brief period it is possible to mark a pool of cells that were in S phase while the BrdU was present. These cells can then be tracked through the remainder of the cell cycle and into the next round of replication, permitting the duration of the cell cycle phases to be determined without the need to induce a potentially toxic cell cycle block. It is also possible to determine and correlate the expression of both internal and external proteins during subsequent stages of the cell cycle. These can be used to further refine the assignment of cell cycle stage or assess effects on other cellular functions such as checkpoint activation or cell death.

Esta metodologia pode ser utilizada para obter uma gama de informações. Alguns aplicativos são descritas aqui. Avaliação da duração do ciclo celular Para determinar o tempo necessário para que as células de trânsito através do ciclo celular, as células são colhidas em vários pontos de tempo após o pulso de BrdU. Os intervalos entre as avaliações pode ser adaptado para as células particulares a serem analisadas. Linhas de células hematopoiética...

Watch this Scientific Journal Video about Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization at JoVE.com

Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization | Biology | Video

Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization

Este protocolo descreve o uso de bromodesoxiuridina (BrdU) para permitir a absorção de seguimento temporais de células que estavam na fase S de um determinado ponto no tempo. A adição de corantes de ADN e rotulagem anticorpo facilita a análise detalhada do destino das células em fase S em momentos posteriores.

Acompanhamento Temporal de progressão do ciclo celular utilizando citometria de fluxo, sem a necessidade de sincronização

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell ...

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Cell Cycle and Division

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19.0K visualizações.  Westmead Millennium Institute for Medical Research and University of Sydney.  Este protocolo descreve o uso de bromodesoxiuridina (BrdU) para permitir a absorção de seguimento temporais de células que estavam na fase S de um determinado ponto no tempo. A adição de corantes de ADN e rotulagem anticorpo facilita a análise detalhada do destino das células em fase S em momentos posteriores. 

Vídeo: Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

Purificação de Citometria de Fluxo de células de camundongo meiótica

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Biologia

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 ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

Optimizado coloração e Proliferação Métodos de Modelagem para Monitoramento Divisão Celular usando Corantes Rastreamento celulares

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

Rastreamento de celular utilizando proteínas Photoconvertible Durante o Desenvolvimento Zebrafish

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal&#8211;to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes.

Many different methods exist for the measurement of extracellular vesicles (EVs) using flow cytometry (FCM). Several aspects should be considered when determining the most appropriate method to use. Two protocols for measuring EVs are presented, using either individual detection or a bead-based approach.

Técnicas de Análise de vesículas extracelulares utilizando citometria de fluxo

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help ...

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

The cell cycle is important for growth, genome replication, and development in all cells. In bacteria, studies of the cell cycle have focused largely on unsynchronized cells making it difficult to order the temporal events required for cell cycle progression, genome replication, and division. Caulobacter crescentus provides an excellent model system for the bacterial cell cycle whereby cells can be rapidly synchronized in a G0 state by density centrifugation. Cell cycle synchronization experiments have been used to establish the molecular events governing chromosome replication and segregation, to map a genetic regulatory network controlling cell cycle progression, and to identify the establishment of polar signaling complexes required for asymmetric cell division. Here we provide a detailed protocol for the rapid synchronization of Caulobacter NA1000 cells. Synchronization can be performed in a large-scale format for gene expression profiling and western blot assays, as well as a small-scale format for microscopy or FACS assays. The rapid synchronizability and high cell yields of Caulobacter make this organism a powerful model system for studies of the bacterial cell cycle.

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

Synchronization of bacterial cells is essential for studies of the bacterial cell cycle and development. Caulobacter crescentus is synchronizable through density centrifugation allowing a rapid and powerful tool for studies of the bacterial cell cycle. Here we provide a detailed protocol for the synchronization of Caulobacter cells.

Sincronização de<em&gt; Caulobacter crescentus</em&gt; Para Investigação do Ciclo Celular Bacteriana

The cell cycle is important for growth, genome replication, and development in all cells.  In bacteria, studies of the cell cycle have focused largely on ...

LED Thermo Flow &#8212; Combining Optogenetics with Flow Cytometry

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the controlled delivery of light to the cell sample and hence the most popular tools for optogenetic studies are microscopy-based cell analyses and in vitro experiments. The flow cytometer has major advantages over a microscope, including the ability to rapidly measure thousands of cells at single cell resolution. However, it is not yet widely used in optogenetics. Here, we present a device that combines the power of optogenetics and flow cytometry: the LED Thermo Flow. This device illuminates cells at specific wavelengths, light intensities and temperatures during flow cytometric measurements. It can be built at low cost and be used with most common flow cytometers. To demonstrate its utility, we characterized the photoswitching kinetics of Dronpa proteins in vivo and in real time. This protocol can be adapted to almost all optically controlled substances and substantially expands the set of possible experiments. More importantly, it will greatly simplify the discovery and development of new optogenetic tools.

LED Thermo Flow &amp;#8212; Combining Optogenetics with Flow Cytometry

Optically controlled substances are powerful tools to study signaling pathways. To expand the spectrum of possible experiments, we developed a device for studying optically controlled substances in real time using flow cytometry: the LED Thermo Flow.

LED Thermo Flow - Combinando optogenética com Citometria de Fluxo

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the ...

Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using Flow Cytometry

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these multi-spanning transmembrane proteins, with or without change in channel gating, is often postulated to contribute significantly in this process. It has thus become critical to develop a method to quantify the change of the relative cell surface expression of cardiac ion channels on a large scale. Herein, a detailed protocol is provided to determine the relative total and cell surface expression of cardiac L-type calcium channels CaV1.2 and membrane-associated subunits in tsA-201 cells using two-color fluorescent cytometry assays. Compared with other microscopy-based or immunoblotting-based qualitative methods, flow cytometry experiments are fast, reproducible, and large-volume assays that deliver quantifiable end-points on large samples of live cells (ranging from 104 to 106 cells) with similar cellular characteristics in a single flow. Constructs were designed to constitutively express mCherry at the intracellular C-terminus (thus allowing a rapid assessment of the total protein expression) and express an extracellular-facing hemagglutinin (HA) epitope to estimate the cell surface expression of membrane proteins using an anti-HA fluorescence conjugated antibody. To avoid false negative, experiments were also conducted in permeabilized cells to confirm the accessibility and proper expression of the HA epitope. The detailed procedure provides: (1) design of tagged DNA (deoxyribonucleic acid) constructs, (2) lipid-mediated transfection of constructs in tsA-201 cells, (3) culture, harvest, and staining of non-permeabilized and permeabilized cells, and (4) acquisition and analysis of fluorescent signals. Additionally, the basic principles of flow cytometry are explained and the experimental design, including the choice of fluorophores, titration of the HA antibody and control experiments, is thoroughly discussed. This specific approach offers objective relative quantification of the total and cell surface expression of ion channels that can be extended to study ion pumps and plasma membrane transporters.

Inherited cardiac arrhythmias are often caused by mutations that alter the surface delivery of one or more ion channels. Here, we adapt flow cytometry assays to provide a quantification of the relative total and cell surface protein expression of recombinant ion channels expressed in tsA-201 cells.

Determinação da superfície celular Relativa e Expressão total de canais recombinante de iões utilizando Citometria de Fluxo

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these ...

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

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

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

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

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

Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The synchronization of cell populations at specific stages of the cell cycle has been found to be very useful in such experimental endeavors. Synchronization of cells by treatment with chemicals that are relatively less toxic can be advantageous over the use of pharmacological inhibitory drugs for the study of consequent cell cycle events and to obtain specific enrichment of selected mitotic stages. Here, we describe the protocol for synchronizing human cells at different stages of the cell cycle, including both in S phase and M phase with a double thymidine block and release procedure for studying the functionality of mitotic proteins in chromosome alignment and segregation. This protocol has been extremely useful for studying the mitotic roles of multifunctional proteins which possess established interphase functions. In our case, the mitotic role of Cdt1, a protein critical for replication origin licensing in G1 phase, can be studied effectively only when G2/M-specific Cdt1 can be depleted. We describe the detailed protocol for depletion of G2/M-specific Cdt1 using double thymidine synchronization. We also explain the protocol of cell fixation, and live cell imaging using high resolution confocal microscopy after thymidine release. The method is also useful for analyzing the function of mitotic proteins under both physiological and perturbed conditions such as for Hec1, a component of the Ndc80 complex, as it enables one to obtain large sample sizes of mitotic cells for fixed and live cell analysis as we show here.&#160;&#160;

We present a protocol for double thymidine synchronization of HeLa cells followed by analysis using high resolution confocal microscopy. This method is key to obtaining large number of cells that proceed synchronously from S phase to mitosis, enabling studies on mitotic roles of multifunctional proteins which also possess interphase functions.

Combinando células mitóticas sincronização e microscopia Confocal de alta resolução para estudar o papel das proteínas Multifunctional ciclo celular durante a mitose

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The ...