Os autores são apoiados pelo Conselho de Pesquisa sueco, a fundação sueca para pesquisas estratégicas, a sociedade de câncer sueco, a sociedade do câncer sueco criança, Wibergs Åke fundação e fundamento Jeanssons.

1. Introduzir a sonda para células <ol><li> Co-transfecção de células com uma sonda FRET-based e uma resistência plasmídeo conferir. Escolha um método de transfecção que seja eficiente na sua cela tipo de interesse. Para as células U2OS, padrão de cálcio métodos de transfecção fosfato dão resultados adequados 15. </li><li> Selecione as células do antibiótico apropriado para pelo menos sete dias. Isso enriquece a quantidade de células que expressam a sonda e limita a quantidade de células com níveis de expressão tóxicos, ou níveis de expressão que afetam gravemente o ciclo celular. </li><li> (Opcional) Selecione para clones estáveis. </li><li> Usando células fixas ou viver, verifique se ambos PCP e YFP estão presentes em todas as células, aproximadamente a mesma proporção. </li><li> No caso de algumas células contêm apenas PCP ou YFP, a recombinação tem provavelmente ocorreu. Recombinação pode ocorrer tanto durante o crescimento de bactérias ou após transfecção da sonda, podendo neste último caso dependem da qualidade plasmídeo. Repita p plasmídeoreparação e linearizar o plasmídeo antes de transfecção se o problema persiste. </li></ol> 2. Raciométrica monitoramento FRET <ol><li> Células de semente em pratos de vidro de fundo, ou lamínulas para montagem em uma câmara. Certifique-se que pratos / lamínulas há 1,5 (170 mm de espessura). </li><li> Montar células em meio de crescimento sem vermelho de fenol em um microscópio de epifluorescência motorizada com controle de temperatura definida para 37 ° C. Garantir a regulação do pH dos meios de comunicação, seja por CO 2 ou usando CO 2 independente de mídia, como Liebowitz-15. </li><li> Concentre-se em células usando luz transmitida. Não olhe nas células usando luz fluorescente. </li><li> Comece por adquirir uma imagem de 12 bits ou 16 usando excitação YFP (YFPex) e YFP emissão (YFPem) filtros e um espelho adequado dicróicas. Use binning máxima disponível que ainda permite a resolução necessária subcelulares. Usando um binning alta para diminuir o tempo de exposição ou a intensidade é crucial para evitar fototoxicidade. </li><li> Measure ou estimar a intensidade do ruído de fundo e aproximada, medindo ou estimando a média aproximada, valores mínimo e máximo de pixels em uma área desprovida de células. Alterar o tempo de exposição e filtros de densidade neutra para que o sinal média na célula é de aproximadamente a intensidade de fundo mais cinco a dez vezes a diferença entre Max e intensidades de fundo min. </li><li> Verifique se as imagens não estão perto de ser saturado. A intensidade máxima na imagem deve ser inferior a metade da faixa dinâmica (por exemplo, max de 2000, em uma imagem de 12 bits) para permitir diferenças de intensidade quando as células entram mitose. </li><li> Repita o passo 2,4-2,6 usando excitação PCP e filtros de emissão YFP e um espelho adequado dicróicas. </li><li> Selecione várias regiões com células transfectadas, excluindo as regiões utilizadas para o ponto 2.4 a 2.7, e garantir que a intensidade máxima é inferior a metade da faixa dinâmica em todas as células. </li><li> Iniciar uma experiência de lapso de tempo adquirir duas imagens a cada 45 min usando YFPex - YFPem e CFPex - define YFPem filtro. </li></ol> 3. Verificação e otimização das condições de <ol><li> Abra as imagens adquiridas e monitorar o tempo do ciclo celular da mitose a mitose de células que expressam a sonda transfectadas. </li><li> Compare o tempo de ciclo celular medidos com os dados publicados para o tipo de célula utilizada. Alternativamente, o filme untransfected células através da aquisição de uma imagem de luz transmitida apenas (por exemplo, DIC ou de contraste de fase) a cada hora para obter tempos de referência do ciclo celular. </li><li> No caso de o tempo de mitose a mitose corresponde à referência timings ciclo celular, vá para a etapa 4. Alternativamente, re-adquirir imagens usando mais difícil de exposição, mais tempo ou vários pontos de z-níveis e repita o passo 3. </li><li> No caso de o tempo de mitose a mitose não corresponde à referência timings do ciclo celular, o filme transfectadas células através da aquisição de uma imagem de luz transmitida apenas (por exemplo, DIC ou de contraste de fase) a cada hora seguido por uma imagem no finaldo experimento para identificar células transfectadas. </li><li> Se as células transfectadas ainda mostram os tempos de ciclo mais longo de células, repita o passo 1, mas transfectar menos sonda ou selecione para clones estáveis ​​que contêm quantidades muito baixas de sonda. </li><li> Se as células transfectadas mostram os tempos de ciclo celular normal, na ausência de luz fluorescente, repita o passo 2, mas menor exposição modificando binning, filtros de densidade neutra, tempo de exposição eo tempo entre as imagens. </li></ol> 4. Analisando FRET <ol><li> A análise pode ser realizada pela maioria dos software dedicado à microscopia. Aqui nós descrevemos como realizar a análise utilizando o ImageJ freeware ( <a href="http://rsb.info.nih.gov/ij" target="_blank">http://rsb.info.nih.gov/ij</a> ) utilizando o plugin Relação Plus ( <a href="http://imagej.nih.gov/ij/plugins/ratio-plus.html" target="_blank">http://imagej.nih.gov/ij/plugins/ratio-plus . html</a> ). </li><li> Abra as imagens no ImageJ. No caso suas imagens estão no formato de um Stac multicolork (por exemplo, Deltavision), separar os canais individuais pelo primeiro convertê-los para um hyperstack: Image-hyperstack-stack para hyperstack e posteriormente dividi-los com pilhas de canal único usando: Image-hyperstack-reduzir a dimensionalidade </li><li> Multiplique o YFPex - pilha YFPem com 3 usando: Processo de Math-Multiply Este passo é opcional, pois se destina apenas a aumentar a clareza visual das imagens, garantindo que os rácios não estão na faixa entre 0 e 1. </li><li> No YFPex - pilha YFPem, desenhe uma região de interesse (ROI) em uma área que é desprovida de células, mas que está perto de uma célula a ser medido. Células diferentes na mesma imagem pode exigir diferentes regiões, tanto como de fundo e intensidade do sinal normalmente é mais forte no centro de uma imagem. </li><li> Adicione o ROI de ROI manager: Analise-tools-ROI gerente. </li><li> Medir a intensidade média do ROI em ambos os YFPex - YFPem eo CFPex - pilha YFPem: Analisar-Medida </em&gt;. </li><li> Repita em intervalo de tempo múltiplos e verificar que as intensidades de fundo perto da célula de interesse são semelhantes ao longo do filme. </li><li> Definir medidas para incluir a intensidade mínima: Analise-Set medições-Min e Max valor de cinza. </li><li> Desenhar uma região de interesse cobrindo a maior parte de uma célula e medir a intensidade mínima em ambos os YFPex - YFPem eo CFPex - pilha YFPem. Pegue a diferença entre a intensidade medida mínima e da intensidade de fundo de 4,6 e dividir por dois. Isso fornece uma estimativa de partida para um valor de corte. </li><li> Além disso Relação aberta. Selecione o YFPex - YFPem eo CFPex - pilha YFPem. Para FRET relação, selecione CFPex - YFPem como a pilha 1, para invertida FRET relação visualizar sondas FRET onde diminui a eficiência sobre a fosforilação, selecione YFPex - YFPem como a pilha 1. </li><li> Inserir as intensidades de fundo medida e os valores de corte. </li><li> Definir o dimensionamento da pilha relação resultante e aplicar um olhar adequado up table (LUT) para visualizar mudanças de relação. </li></ol> 5. Resultados representante Plk1 primeira atividade é visível no núcleo em fase G2 e picos durante a mitose. A Figura 3 mostra um experimento usando as configurações fototoxicidade mínima, conforme descrito na seção 2. Por favor note que este é um resultado representativo inicial e que as condições de exposição ou o tempo entre as imagens pode ser modificado para aumentar a relação sinal-ruído ou a resolução temporal. Mostra a figura 3D que a maioria das células que expressam a sonda proliferam com um tempo de ciclo celular entre 20 e 25 horas, indicando que as condições de imagem e níveis de expressão da sonda não afetam timings ciclo celular. Embora não haja ruído considerável, a tendência de Plk1 atividade crescente no G2 e mitose atingindo o pico em 14 é claramente visível (fig. 3A). Processamento dos dados brutos, aqui pela média filtragem apresentado como um quimógrafo (fig. 3B), ou quantificação do ra média invertida<html> tio (fig. 3C) pode melhorar a clareza. <img alt="Figura 1" src="/files/ftp_upload/3410/3410fig1.jpg" /> Figura 1. Princípio de uma sonda FRET baseado para monitorar as atividades quinase e fosfatase. Dois fluoróforos, tipicamente PCP (azul) e YFP (verde), são conectados por um domínio fosfo-binding (laranja) e uma seqüência fosforilável (amarelo). Fosforilação (vermelho) a mediação obrigatória para o domínio fosfo-binding, induzindo uma mudança conformacional na sonda. O conformacional alteração resulta em uma diferença na distância ou orientação entre os dois fluoróforos, que afeta o FRET eficiência entre PCP e YFP. FRET pode ser visualizado pelo PCP emocionante e de monitorização das emissões YFP (linhas pontilhadas). <img alt="Figura 2" src="/files/ftp_upload/3410/3410fig2.jpg" /> Figura 2. Esboço esquemático do procedimento experimental. p_upload/3410/3410fig3.jpg &quot;/&gt; Figura 3. Plk1 atividade é o primeiro visível no núcleo em fase G2 e picos durante a mitose. U2OS células expressando um FRET baseado sonda de monitoramento de atividade Plk1 9,14 foram filmados por 60 horas usando um Deltavision Spectris Sistema de imagens equipado com um objetivo 20x de ar NA 0,7 e uma lâmpada de mercúrio. Condições imagens foram selecionadas para causar fototoxicidade mínima, conforme descrito na seção 2, usando binning 4x e filtros de densidade neutra que 99% de bloco da luz recebida. A, representação de cores falsas de FRET relação invertida, seguindo uma célula através de 4 divisões. B, quimógrafo da célula mostrada na A depois de aplicar um filtro de média. C, quantificação do invertida FRET relação entre a célula mostrada na A. D, entrada mitótica acumulado de 50 FRET-sonda células que expressam, incluindo divisões de células-filhas.

<ol>
	<li>Sun, Y., Wallrabe, H., Seo, S. A., Periasamy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FRET+microscopy+in+2010:+the+legacy+of+Theodor+Forster+on+the+100th+anniversary+of+his+birth.">FRET microscopy in 2010: the legacy of Theodor Forster on the 100th anniversary of his birth.</a> Chemphyschem. 12, 462-474 (2011).</li><li>Allen, M. D., Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+dynamics+of+protein+kinase+A+activity+visualized+by+FRET-based+reporters.">Subcellular dynamics of protein kinase A activity visualized by FRET-based reporters.</a> Biochem. Biophys. Res. Commun. 348, 716-721 (2006).</li><li>Kunkel, M. T., Ni, Q., Tsien, R. Y., Zhang, J., Newton, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatio-temporal+dynamics+of+protein+kinase+B/Akt+signaling+revealed+by+a+genetically+encoded+fluorescent+reporter.">Spatio-temporal dynamics of protein kinase B/Akt signaling revealed by a genetically encoded fluorescent reporter.</a> J. Biol. Chem. 280, (2005).</li><li>Violin, J. D., Zhang, J., Tsien, R. Y., Newton, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetically+encoded+fluorescent+reporter+reveals+oscillatory+phosphorylation+by+protein+kinase.">A genetically encoded fluorescent reporter reveals oscillatory phosphorylation by protein kinase.</a> C. J. Cell. Biol. 161, 899-909 (2003).</li><li>Kunkel, M. T., Toker, A., Tsien, R. Y., Newton, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-dependent+regulation+of+protein+kinase+D+revealed+by+a+genetically+encoded+kinase+activity+reporter.">Calcium-dependent regulation of protein kinase D revealed by a genetically encoded kinase activity reporter.</a> Journal of Biological Chemistry. 282, 6733-6742 (2007).</li><li>Harvey, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetically+encoded+fluorescent+sensor+of+ERK+activity.">A genetically encoded fluorescent sensor of ERK activity.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 19264-19269 (2008).</li><li>Fosbrink, M., Aye-Han, N. N., Cheong, R., Levchenko, A., Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+JNK+activity+dynamics+with+a+genetically+encoded+fluorescent+biosensor.">Visualization of JNK activity dynamics with a genetically encoded fluorescent biosensor.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 5459-5464 (2010).</li><li>Gavet, O., Pines, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+activation+of+CyclinB1-Cdk1+coordinates+entry+to+mitosis.">Progressive activation of CyclinB1-Cdk1 coordinates entry to mitosis.</a> Dev. Cell. 18, 533-543 (2010).</li><li>Fuller, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Midzone+activation+of+aurora+B+in+anaphase+produces+an+intracellular+phosphorylation+gradient.">Midzone activation of aurora B in anaphase produces an intracellular phosphorylation gradient.</a> Nature. 453, 1132-1136 (2008).</li><li>Ni, Q., Titov, D. V., Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+protein+kinase+dynamics+in+living+cells+with+FRET+reporters.">Analyzing protein kinase dynamics in living cells with FRET reporters.</a> Methods. 40, 279-286 .</li><li>Morgan, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The cell cycle : principles of control. , (2007).</li><li>Lindqvist, A., Rodriguez-Bravo, V., Medema, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+decision+to+enter+mitosis:+feedback+and+redundancy+in+the+mitotic+entry+network.">The decision to enter mitosis: feedback and redundancy in the mitotic entry network.</a> J. Cell. Biol. 185, 193-202 (2009).</li><li>Gavet, O., Pines, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+cyclin+B1-Cdk1+synchronizes+events+in+the+nucleus+and+the+cytoplasm+at+mitosis.">Activation of cyclin B1-Cdk1 synchronizes events in the nucleus and the cytoplasm at mitosis.</a> J. Cell. Biol. 189, 247-259 (2010).</li><li>Macurek, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polo-like+kinase-1+is+activated+by+aurora+A+to+promote+checkpoint+recovery.">Polo-like kinase-1 is activated by aurora A to promote checkpoint recovery.</a> Nature. 455, 119-123 (2008).</li><li>van der Eb, A. J., Graham, F. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assay+of+transforming+activity+of+tumor+virus+DNA.">Assay of transforming activity of tumor virus DNA.</a> Methods. Enzymol. 65, 826-839 (1980).</li><li>Lamprecht, M. R., Sabatini, D. M., Carpenter, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CellProfiler:+free,+versatile+software+for+automated+biological+image+analysis.">CellProfiler: free, versatile software for automated biological image analysis.</a> Biotechniques. 42, 71-75 (2007).</li><li>Roszik, J., Lisboa, D., Szollosi, J., Vereb, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+intensity-based+ratiometric+FRET+in+image+cytometry--approaches+and+a+software+solution.">Evaluation of intensity-based ratiometric FRET in image cytometry--approaches and a software solution.</a> Cytometry A. 75, 761-767 (2009).</li></ol>

Não temos nada a revelar.

Monitoramento FRET todo o ciclo celular requer considerações que são menos cruciais na avaliação de curto prazo respostas a estímulos externos. Primeiro, a progressão do ciclo celular é facilmente perturbado por sinalização stress, exigindo que fototoxicidade é reduzido ao mínimo. Segundo, todos os repórteres podem afetar os processos celulares pela titulação fora quinases, fosfatases ou domínios de interação. A maneira provavelmente mais simples de avaliar se as condições experimentais são adequado...

<table border="1"><tbody><tr><td> Reagente </td><td> Número Catálogo </td><td> Companhia </td></tr><tr><td> Leibovitz L-15, sem vermelho de fenol </td><td> 21083-027 </td><td> GIBCO, pela Life Technologies </td></tr><tr><td> DMEM + Glutamax-I </td><td> 31966 </td><td> GIBCO, pela Life Technologies </td></tr><tr><td> De soro fetal bovino (FBS) </td><td> SV30160.03 </td><td> HyClone </td></tr><tr><td> 0,05% Tripsina EDTA </td><td> SH30236.01 </td><td> HyClone </td></tr><tr><td> Penicilina-estreptomicina </td><td> SV30010 </td><td> HyClone </td></tr><tr><td> DPBS </td><td> 14287 </td><td> GIBCO, pela Life Technologies </td></tr><tr><td> Puromicina </td><td> P8833 </td><td> Sigma-Aldrich </td></tr></tbody></table>

monitoring kinase and phosphatase activities through the cell cycle by ratiometric fret

Förster transferência de energia por ressonância (FRET) baseado em uma repórteres permitir a avaliação da quinase endógena e atividades da fosfatase em células vivas. Sondas tais tipicamente consistem de variantes do PCP e YFP, interveio por uma seqüência fosforilável e um domínio fosfo-binding. Após a fosforilação, a sonda conformação mudanças, o que resulta em uma mudança da distância ou da orientação entre PCP e YFP, levando a uma mudança na eficiência FRET (Fig. 1). Sondas de vários trabalhos foram publicados na última década, o monitoramento do equilíbrio atividade de quinases e fosfatases múltiplas, incluindo repórteres de PKA 2, PKB 3, PKC 4, PKD 5, 6 ERK, JNK 7, CDK 18, Aurora B 9 e 9 Plk1 . Dado o design modular, sondas adicionais é provável que surjam no futuro próximo 10. Progressão do ciclo celular é afetada pelo estresse signaling caminhos 11. Notavelmente, o ciclo celular é regulado de forma diferente durante imperturbável crescimento em relação a quando as células estão se recuperando do estresse 12. Time-lapse de imagens de células através do ciclo celular, portanto, requer um cuidado especial. Isso se torna um problema particularmente quando se emprega imagens raciométrica, uma vez que duas imagens com uma alta relação sinal-ruído são obrigados a interpretar corretamente os resultados. Raciométrica FRET imagem das mudanças do ciclo celular dependentes em atividades quinase e fosfatase tem sido predominantemente restrita ao sub-seções do ciclo celular 8,9,13,14. Aqui, discutimos um método para monitorar FRET baseado em sondas utilizando imagens raciométrica todo o ciclo celular humano. O método baseia-se em equipamento que está disponível para muitos pesquisadores em ciências da vida e não requer conhecimento especializado de microscopia ou processamento de imagem.

Watch this Scientific Journal Video about Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET at JoVE.com

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

FRET baseado jornalistas estão cada vez mais utilizados para monitorar atividades quinase e fosfatase em células vivas. Aqui nós descrevemos um método de como usar FRET baseado jornalistas para avaliar o ciclo de dependentes de células alterações na fosforilação alvo.

Atividades de monitoramento quinases e fosfatases através do ciclo celular por raciométrica FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

monitoring-kinase-phosphatase-activities-through-cell-cycle

Research

JoVE Journal

Biology

14.2K visualizações.  Karolinska Institutet. FRET baseado jornalistas estão cada vez mais utilizados para monitorar atividades quinase e fosfatase em células vivas. Aqui nós descrevemos um método de como usar FRET baseado jornalistas para avaliar o ciclo de dependentes de células alterações na fosforilação alvo.

Vídeo: Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

Drawing Blood from Rats through the Saphenous Vein and by Cardiac Puncture

Drawing blood from rodents is necessary for a large number of both in vitro and in vivo studies. Sites of blood draws are numerous in rodents: retro-orbital sinus, jugular vein, maxillary vein, saphenous vein, heart. Each technique has its advantages and disadvantages, and some are not approved any more in some countries (e.g., retro-orbital draws in Holland). A discussion of different techniques for drawing blood are available 1-3.
Here, we present two techniques for drawing blood from rats, each with its specific applications.
Blood draw from the saphenous vein, provided it is done properly, induces minimal distress in animals and does not require anesthesia. This technique allows repeated draws of small amounts of blood, such as needed for pharmacokinetic studies 4,5, determining plasma chemistry, or blood counts 6.
Cardiac puncture allows the collection of large amounts of blood from a single animal (up to 10 ml of blood can be drawn from a 150 g rat). This technique is therefore very useful as a terminal procedure when drawing blood from the saphenous would not provide a large enough sample. We use cardiac puncture when we need sufficient amounts of serum from a specific strain of rats to grow T lymphocyte lines in vitro 4-9.

Blood draws are necessary in a large number of studies, for example to study the pharmacokinetics profile of a compound. Here, we demonstrate how to draw blood from rats using two techniques: blood draw from the saphenous vein or by cardiac puncture.

Desenho de sangue de ratos através da veia safena e por punção cardíaca

Drawing blood from rodents is necessary for a large number of both in vitro and in vivo studies. Sites of blood draws are numerous in rodents: ...

Biologia

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 ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

FRET Microscopia para Monitoramento em Tempo Real de eventos de sinalização em células vivas utilizando biossensores unimolecular

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

Studying DNA Looping by Single-Molecule FRET

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into nucleosomes. Thermodynamics of dsDNA bending has been studied by a method called cyclization which relies on DNA ligase to covalently join short sticky ends of a dsDNA. However, ligation efficiency can be affected by many factors that are not related to dsDNA looping such as the DNA structure surrounding the joined sticky ends, and ligase can also affect the apparent looping rate through mechanisms such as nonspecific binding. Here, we show how to measure dsDNA looping kinetics without ligase by detecting transient DNA loop formation by FRET (Fluorescence Resonance Energy Transfer). dsDNA molecules are constructed using a simple PCR-based protocol with a FRET pair and a biotin linker. The looping probability density known as the J factor is extracted from the looping rate and the annealing rate between two disconnected sticky ends. By testing two dsDNAs with different intrinsic curvatures, we show that the J factor is sensitive to the intrinsic shape of the dsDNA.

This study presents a detailed experimental procedure to measure looping dynamics of double-stranded DNA using single-molecule Fluorescence Resonance Energy Transfer (FRET). The protocol also describes how to extract the looping probability density called the J factor.

Estudar DNA Looping por Single-Molecule FRET

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into ...

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 ...

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.

This protocol describes the use of bromodeoxyuridine (BrdU) uptake to permit the temporal tracking of cells that were in S phase at a specific point in time. Addition of DNA dyes and antibody labeling facilitates detailed analysis of the fate of the S phase cells at later times.

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 ...

Experimental Approaches to Study Mitochondrial Localization and Function of a Nuclear Cell Cycle Kinase, Cdk1

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The nuclear-encoded proteins are transported into mitochondria guided by their mitochondria targeting sequences (MTS); however, a majority of mitochondrial localized proteins lack an identifiable MTS. Nevertheless, the fact that MTS can instruct proteins to go into the mitochondria provides a valuable tool for studying mitochondrial functions of normally nuclear and/or cytoplasmic proteins. We have recently identified the cell cycle kinase CyclinB1/Cdk1 complex in the mitochondria. To specifically study the mitochondrial functions of this complex, mitochondrial overexpression and knock-down of this complex without interfering with its nuclear or cytoplasmic functions were essential. By tagging CyclinB1/Cdk1 with MTS, we were able to achieve mitochondrial overexpression of this complex to study its mitochondrial targets as well as functions. Via tagging dominant-negative Cdk1 with MTS, inhibition of Cdk1 activity was accomplished particularly in the mitochondria. Potential mitochondrial targets of CyclinB1/Cdk1 complex were identified using a gel-based proteomics approach. Unlike traditional 2D gel analysis, we employed 2-dimensional difference gel electrophoresis (2D-DIGE) technology followed by phosphoprotein staining to fluorescently label differentially phosphorylated proteins in mitochondrial Cdk1 expressing cells. Identification of phosphoprotein spots that were altered in wild type versus dominant negative Cdk1 bearing mitochondria revealed the identity of mitochondrial targets of Cdk1. Finally, to determine the effect of CyclinB1/Cdk1 mitochondrial localization in cell cycle progression, a cell proliferation assay using a synthetic thymidine analogue EdU (5-ethynyl-2&#8242;-deoxyuridine) was used to monitor the cells as they go through the cell cycle and replicate their DNA. Altogether, we demonstrated a variety of approaches available to study mitochondrial localization and activity of a cell cycle kinase. These are advanced, yet easy to follow methods that will be beneficial to many cell biology researchers.

Here, we outline how to study mitochondrial localization of a (cell cycle) kinase, and how to determine its sub-mitochondrial location as well as potential mitochondrial substrates/targets. Forced expression of proteins into the mitochondria provides a useful tool for studying the functional consequences of mitochondrial localization of a protein of interest.

Abordagens experimentais para estudar mitocondrial Localização e função de um ciclo celular Nuclear Kinase, Cdk1

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Fecal Análise de glicocorticóides: Non-invasive Monitoring Adrenal em eqüídeos

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

G Protein-selective GPCR Conformations Measured Using FRET Sensors in a Live Cell Suspension Fluorometer Assay

F&#1255;rster resonance energy transfer (FRET)-based studies have become increasingly common in the investigation of GPCR signaling. Our research group developed an intra-molecular FRET sensor to detect the interaction between G&#945; subunits and GPCRs in live cells following agonist stimulation. Here, we detail the protocol for detecting changes in FRET between the &#946;2-adrenergic receptor and the G&#945;s C-terminus peptide upon treatment with 100 &#181;M isoproterenol hydrochloride as previously characterized1. Our FRET sensor is a single polypeptide consisting serially of a full-length GPCR, a FRET acceptor fluorophore (mCitrine), an ER/K SPASM (systematic protein affinity strength modulation) linker, a FRET donor fluorophore (mCerulean), and a G&#945; C-terminal peptide. This protocol will detail cell preparation, transfection conditions, equipment setup, assay execution, and data analysis. This experimental design detects small changes in FRET indicative of protein-protein interactions, and can also be used to compare the strength of interaction across ligands and GPCR-G protein pairings. To enhance the signal-to-noise in our measurements, this protocol requires heightened precision in all steps, and is presented here to enable reproducible execution.

Simple methods to detect the selective activation of G proteins by G protein-coupled receptors remain an outstanding challenge in cell signaling. Here, F&#1255;rster resonance energy transfer (FRET) biosensors have been developed by pairwise tethering a GPCR to G protein peptides to probe conformational changes at controlled concentrations in live cells.

Conformações GPCR G Protein selectivos medida usando FRET Sensores em um Fluorometer Ensaio de células vivas de suspensão

F&#1255;rster resonance energy transfer (FRET)-based studies have become increasingly common in the investigation of GPCR signaling. Our research group ...

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 ...