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).

El protocolo descrito aquí utiliza la línea celular de leucemia linfoblástica aguda NALM6 pero se puede aplicar a otros tipos de células. 1. Soluciones y Reactivos <ol><li> RPMI completo <ol><li> Añadir 56 ml de suero de ternera fetal (FCS) y 5,5 ml de 200 mM de L-glutamina a una botella de 500 ml de medio RPMI-1640. </li></ol></li><li> BrdU de la Solución <ol><li> Preparar 32,5 mM BrdU (10 mg / ml) en fosfato de Dulbecco Buffered Saline (DPBS). </li></ol></li><li> BrdU completo RPMI <ol><li> Añadir 6,2 l de solución madre de BrdU a 10 ml de RPMI completo. </li></ol></li><li> Solución DNasa <ol><li> Preparar 1 mg de DNasa / ml en DPBS. </li></ol></li><li> La tinción Buffer <ol><li> Preparar 3% de FCS inactivado por calor y 0,09% de azida de sodio en DPBS. </li></ol></li><li> Consulte la Lista de materiales para las definiciones de Fijación Buffer, permeabilización Buffer y el tampón de lavado. </li></ol> 2. Las células NoTe: Las células no fueron cultivadas por más de 6 meses. Este método es directamente adaptable a cualquier línea celular no adherente con ajustes a la densidad de células y medios de cultivo. Utilice células que están creciendo exponencialmente a la iniciación del experimento. <ol><li> Mantener células NALM6 en frascos de cultivo T-75 en RPMI completo. Realice todos los pasos en condiciones estériles utilizando una Clase II Bioseguridad gabinete. <ol><li> Mantener células NALM6 entre 2.1 x 10 6 células por ml mediante el fraccionamiento de la cultura tres veces por semana. </li><li> Se incuba a 37 ° C en 5% de CO 2 en el aire. </li></ol></li></ol> 3. Pulso Etiquetado de células con BrdU PRECAUCIÓN: Manipule BrdU con cuidado, ya que es un mutágeno potencial y teratógeno. <ol><li> Centrifugar las células a 150 xg durante 5 min. Nota: La transferencia de células en medio fresco mejora la reproducibilidad de los resultados. </li><li> Realizar un recuento de células y las células volver a suspender en RPMI completo a 2 x 10 6 canas / ml. </li><li> Diluir las células en 1 2 con BrdU RPMI completo produciendo una concentración final de células de 1 x 10 6 células / ml. </li><li> Incubar a 37 ° C con 5% de CO2 durante 45 min, luego se diluye células 1 en 10 con RPMI completo. Centrifugar las células a 150 xg durante 5 min y cuidadosamente descartan todo el sobrenadante. </li><li> Resuspender las células en un volumen pequeño (~ 100 l) de RPMI completo, realizar un recuento de células y se ajustan a 1 x 10 6 células / ml. </li><li> Pipetear 1 ml de las células en los pocillos de una placa de 48 pocillos. Pipeta 1 ml de DPBS en cualquier pozo desocupadas para obtener resultados más reproducibles. </li><li> Incubar a 37 ° C en 5% de CO2 en aire para puntos de tiempo deseados, aquí 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23 y 24 h. Nota: La cantidad de tiempo dependerá de lo que tiene como objetivo el diseño experimental para medir. </li><li> Transferencia de todas las células en tubos de FACS utilizando una pipeta. Enjuague la secuencialmente bien con 1 ml volumes de PBS a un volumen total final de 5 ml. </li><li> Centrifugar a 150 xg durante 5 minutos y retirar con cuidado todo el sobrenadante. Las células están listas para la tinción, realizan esta (sección 4) inmediatamente. </li></ol> 4. La tinción celular Nota: Si se requiere tinción de la superficie de las células que realizar antes de la fijación, asegurando que las células se mantienen a 4 ° C durante todo. <ol><li> Resuspender las células en 100 l de tampón de tinción (para la tinción de superficie opcional, añadir el volumen recomendado de anticuerpo a la superficie antígenos y se incuba durante 30 min a 4 ° C). </li><li> Añadir 1 ml de tampón de tinción, de centrifugación durante 5 min a 150 xg y descartar el sobrenadante. Nota: El anticuerpo específico, concentración, tiempo de incubación etc. variará dependiendo de las metas experimentales específicas. </li><li> Fijación y permeabilización <ol><li> Resuspender las células en 100 l de tampón de fijación y se incuba durante 15 min a temperatura ambiente. </li><li> Añadir 1 ml otampón de lavado f, centrifugar durante 5 min a 150 xg y desechar el sobrenadante. </li><li> Resuspender las células en 100 l de tampón de permeabilización e incubar las células durante 10 min en hielo. </li><li> Añadir 1 ml de tampón de lavado, de centrifugación durante 5 min a 150 xg, y descartar el sobrenadante. </li><li> Resuspender las células en 100 l de tampón de fijación por tubo y se incuba durante 5 min a temperatura ambiente. </li><li> Añadir 1 ml de tampón de lavado, de centrifugación durante 5 min a 150 xg, y descartar el sobrenadante. Nota: El protocolo puede ser una pausa aquí, si es necesario. Las células fijadas son estables durante varios días a 4 ° C si se resuspendieron en tampón de tinción. Retire el tampón de tinción después de la centrifugación antes de continuar. </li></ol></li><li> DNasa tratamiento <ol><li> Resuspender las células en 100 l de solución de DNasa (30 g de DNasa / 10 6 células) y se incuban las células durante 1 hora a 37 ° C. </li><li> Añadir 1 ml de tampón de lavado, centrifugar a 150 xg durante 5 min y desechar el sobrenadante. </li&gt; </ol></li><li> Antibody tinción Nota: La tinción para marcadores intracelulares distintos de BrdU se puede realizar simultáneamente con la tinción de BrdU. <ol><li> IMPORTANTE: Preparar los controles de compensación que constan de células no teñidas y células marcadas con cada solo fluorocromo. Lo ideal es utilizar los mismos anticuerpos para los controles de compensación como los utilizados en los tubos experimentales. Sin embargo, si esto no es factible, los anticuerpos a los antígenos sustitutos altamente expresados ​​conjugarse con el mismo fluorocromo. </li><li> Resuspender las células en 50 l de tampón de lavado y añadir 1 l / 10 6 células de anticuerpo BrdU. Nota: anticuerpos conjugado directamente a otros antígenos intracelulares específicas también se pueden añadir. NOTA:. Los anticuerpos frente a la histona H3 fosforilada en Ser10 se pueden utilizar para discriminar entre células en G2 y M, la histona H3 es fosforilada en Ser10 durante la mitosis 10 Los anticuerpos frente a cdc2 fosforilados en Tyr15 puede ser utilizado para detectar las células que have comprometida con la mitosis. 11 </li><li> Se incuban las células durante 20 min a temperatura ambiente. </li><li> Añadir 1 ml de tampón de lavado, las células de centrifugación a 150 xg durante 5 min y desechar el sobrenadante. </li></ol></li></ol><ol start="6"><li> ADN de manchas de Análisis de Ciclo Celular <ol><li> Aflojar pellet y añadir 20 l de la solución de 7-AAD (0,25 g). Nota: Es importante utilizar una cantidad constante de 7-AAD / célula. </li><li> Resuspender las células en 1 ml de tampón de tinción. </li></ol></li></ol> 5. Recogida de Citometría de Flujo de Datos Nota: La máquina requerida dependerá del número y naturaleza de los fluorocromos usados. <ol><li> Recoge los siguientes parámetros: FSC-A, SSC-A, FSC-H (FSC-W se pueden utilizar en lugar de FSC-H) y 7-AAD fluorescencia en una escala lineal. Recoger el canal de APC en una escala logarítmica. Recoge los canales adicionales que se requieren para la evaluación de la superficie o internos etiquetas utilizando una escala logarítmica. </li><li> Realizar comcompensación de las señales en los espectros de emisión observada entre diferentes fluorocromos antes de analizar las muestras superpuestas. Nota: La mayoría de citómetros de flujo realizarán automáticamente. </li><li> Recoge al menos 10.000 eventos en cada muestra. </li></ol> 6. Análisis de Citometría de Flujo de Datos Nota: FlowJo se utilizó en este estudio para el análisis de citometría de flujo de datos, pero otros paquetes de software también pueden ser utilizados. La estrategia gating se ilustra en la Figura 1. <ol><li> Identificar la población de células viables utilizando parámetros FSC-A y SSC-A. </li><li> Dentro de esta población excluir dobletes y agregados utilizando FSC-A y FSC-H (FSC-W también puede ser utilizado aquí). </li><li> Dentro de esta población establecer un gráfico de puntos usando 7-AAD en el eje x y BrdU-APC en el eje y. </li></ol><img alt="Figura 1" src="/files/ftp_upload/52840/52840fig1highres.jpg" width="700" /> Figura 1: Estrategia de apertura de puerta izquierda pa.nel: células ungated se muestran en una FCS-A vs SSC-Un gráfico de puntos. La población de células viable se identifica por la puerta se muestra. Panel central: células cerradas desde el panel de la izquierda se muestran en una FSC-A vs FSC-H gráfico de puntos (FSC-W se puede utilizar en lugar de la altura). Dobletes y agregados se identifican y excluidos por la puerta se muestra. Panel derecho: células cerradas a partir de la fecha de exclusión doblete en el panel central se muestran en un 7-AAD vs. APC-Un gráfico de puntos. El anticuerpo BrdU se etiqueta con APC que permite la identificación de las células que han incorporado BrdU durante el etiquetado pulso. 7-AAD proporciona información sobre el contenido de ADN. La puerta superior define células positivas para BrdU y por lo tanto en la fase S en el momento del impulso de BrdU, la puerta inferior izquierda, las células en G 0/1 y la puerta inferior derecha los de G 2 / M. <a href="https://www.jove.com/files/ftp_upload/52840/52840fig1highres.jpg" target="_blank">Por favor, haga clic aquí para ver una versión más grande de esta cifra.</a> <ol start="4"><li> Ciclo Celular A<html> nálisis <ol><li> Abra la primera archivo de datos y la puerta a las células en la puerta de exclusión doblete. </li><li> Analizar esta población para la distribución del ciclo celular (que se encuentra bajo plataformas de software FloJo) y utilizar el modelo de Dean-Jett-Fox. </li><li> Obtener las posiciones del G 0/1 y G 2 / M picos utilizando crear puertas. </li><li> Puerta en la BrdU células positivas y sujetos a esta población con el mismo análisis del ciclo celular. </li><li> Proporcionar las posiciones para el G 0/1 y G 2 / M picos mediante la aplicación de las mismas puertas de crear puertas y el establecimiento de restricciones (utilizando las puertas creadas) para las posiciones del G 0/1 y G 2 / M picos. Esto se ilustra en los primeros 2 paneles de la Figura 2. Nota: Otro software también se puede utilizar para analizar los datos y las instrucciones podría variar en consecuencia. </li></ol></li></ol>840 / 52840fig2highres.jpg &quot;width =&quot; 700 &quot;/&gt; Figura 2:. Progresión El primer panel de Ciclo Celular (Todas las células) es cerrada en la población de células definida por la puerta de la exclusión doblete. Esta población se muestra en el histograma con 7-AAD en el eje X. El pico de la 0/1 pico G está indicado por la flecha por debajo del eje. En los paneles posteriores células BrdU positivas han sido cerrada en como se muestra en la Figura 1. Se aplica el valor para la posición G 0/1 obtenida cuando gating de la puerta de exclusión doblete a las células BrdU positivas cerradas dentro de software FlowJo ciclo celular. Cada panel posterior fue cerrada en la población BrdU positivo como se muestra en la Figura 1 y la posición del pico G 0/1 basado en el valor obtenido en el análisis de toda la población como se muestra en los dos primeros paneles. Uso de la fracción negativa BrdU para identificar la ubicación de la población G 0/1 para las células BrdU positivas en la misma sample controla de ningún ligeras diferencias en la intensidad de la mancha de ADN entre muestras. El número que aparece en cada panel representa el tiempo desde que terminó el pulso de BrdU. Las fases del ciclo celular calculados se muestran en verde sombra. <a href="https://www.jove.com/files/ftp_upload/52840/52840fig2highres.jpg" target="_blank">Por favor, haga clic aquí para ver una versión más grande de esta figura.</a>

<ol>
	<li>Banfalvi, G., Banfalvi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods Mol Biol. 761, 1-23 (2011).</li><li>Sakaue-Sawano, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+spatiotemporal+dynamics+of+multicellular+cell-cycle+progression.">Visualizing spatiotemporal dynamics of multicellular cell-cycle progression.</a> Cell. 132, 487-498 (2008).</li><li>Harper, J. W., Elledge, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+DNA+damage+response:+ten+years+after.">The DNA damage response: ten years after.</a> Molecular Cell. 28, 739-745 (2007).</li><li>Latt, S. A., George, Y. S., Gray, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+analysis+of+bromodeoxyuridine-substituted+cells+stained+with+33258+Hoechst.">Flow cytometric analysis of bromodeoxyuridine-substituted cells stained with 33258 Hoechst.</a> The Journal of Histochemistry and Cytochemistry. 25, 927-934 (1977).</li><li>Gratzner, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibody+to+5-bromo-+and+5-iododeoxyuridine:+A+new+reagent+for+detection+of+DNA+replication.">Monoclonal antibody to 5-bromo- and 5-iododeoxyuridine: A new reagent for detection of DNA replication.</a> Science. 218, 474-475 (1982).</li><li>Carayon, P., Bord, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+DNA-replicating+lymphocyte+subsets+using+a+new+method+to+label+the+bromo-deoxyuridine+incorporated+into+the+DNA.">Identification of DNA-replicating lymphocyte subsets using a new method to label the bromo-deoxyuridine incorporated into the DNA.</a> Journal of Immunological Methods. 147, 225-230 (1992).</li><li>Gonchoroff, N. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=S-phase+detection+with+an+antibody+to+bromodeoxyuridine.+Role+of+DNase+pretreatment.">S-phase detection with an antibody to bromodeoxyuridine. Role of DNase pretreatment.</a> Journal of Immunological Methods. 93, 97-101 (1986).</li><li>Rabinovitch, P. S., Torres, R. M., Engel, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+cell+cycle+analysis+and+two-color+surface+immunofluorescence+using+7-amino-actinomycin+D+and+single+laser+excitation:+applications+to+study+of+cell+activation+and+the+cell+cycle+of+murine+Ly-1+B+cells.">Simultaneous cell cycle analysis and two-color surface immunofluorescence using 7-amino-actinomycin D and single laser excitation: applications to study of cell activation and the cell cycle of murine Ly-1 B cells.</a> Journal of Immunology. 136, 2769-2775 (1986).</li><li>Saunders, P. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RAD001+(everolimus)+induces+dose-dependent+changes+to+cell+cycle+regulation+and+modifies+the+cell+cycle+response+to+vincristine.">RAD001 (everolimus) induces dose-dependent changes to cell cycle regulation and modifies the cell cycle response to vincristine.</a> Oncogene. 32, 4789-4797 (2013).</li><li>Hendzel, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitosis-specific+phosphorylation+of+histone+H3+initiates+primarily+within+pericentromeric+heterochromatin+during+G2+and+spreads+in+an+ordered+fashion+coincident+with+mitotic+chromosome+condensation.">Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation.</a> Chromosoma. 106, 348-360 (1997).</li><li>Draetta, G., Beach, D. <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+cdc2+protein+kinase+during+mitosis+in+human+cells:+cell+cycle-dependent+phosphorylation+and+subunit+rearrangement.">Activation of cdc2 protein kinase during mitosis in human cells: cell cycle-dependent phosphorylation and subunit rearrangement.</a> Cell. 54, 17-26 (1988).</li><li>Saunders, P. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RAD001+(everolimus)+induces+dose-dependent+changes+to+cell+cycle+regulation+and+modifies+the+cell+cycle+response+to+vincristine.">RAD001 (everolimus) induces dose-dependent changes to cell cycle regulation and modifies the cell cycle response to vincristine.</a> Oncogene. , (2012).</li><li>Knudsen, R. C., Ahmed, A. A., Sell, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+microassay+for+lymphotoxin+using+microculture+plates+and+the+multiple+automated+sample+harvester.">An in vitro microassay for lymphotoxin using microculture plates and the multiple automated sample harvester.</a> Journal of Immunological Methods. 5, 55-63 (1974).</li><li>Beck, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation+kinetics+of+perturbed+cell+populations+determined+by+the+bromodeoxyuridine-33258+technique:+radiotoxic+effects+of+incorporated+[3H]thymidine.">Proliferation kinetics of perturbed cell populations determined by the bromodeoxyuridine-33258 technique: radiotoxic effects of incorporated [3H]thymidine.</a> Cytometry. 2, 170-174 (1981).</li><li>Miller, M. J., Wei, S. H., Parker, I., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+imaging+of+lymphocyte+motility+and+antigen+response+in+intact+lymph+node.">Two-photon imaging of lymphocyte motility and antigen response in intact lymph node.</a> Science. 296, 1869-1873 (2002).</li><li>Collins, J. M., Berry, D. E., Bagwell, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+rates+of+DNA+synthesis+during+the+S+phase+of+log+phase+HeLa+S3,+WI-38,+and+2RA+cells.">Different rates of DNA synthesis during the S phase of log phase HeLa S3, WI-38, and 2RA cells.</a> Journal of Biological Chemistry. 255, 3585-3590 (1980).</li><li>Schwarting, R., Gerdes, J., Niehus, J., Jaeschke, L., Stein, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+the+growth+fraction+in+cell+suspensions+by+flow+cytometry+using+the+monoclonal+antibody+Ki-67.">Determination of the growth fraction in cell suspensions by flow cytometry using the monoclonal antibody Ki-67.</a> Journal of Immunological Methods. 90, 65-70 (1986).</li><li>Danova, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cycle-related+proteins:+a+flow+cytofluorometric+study+in+human+tumors.">Cell cycle-related proteins: a flow cytofluorometric study in human tumors.</a> Biology of the Cell. 64, 23-28 (1988).</li><li>Juan, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+H3+phosphorylation+and+expression+of+cyclins+A+and+B1+measured+in+individual+cells+during+their+progression+through+G2+and+mitosis.">Histone H3 phosphorylation and expression of cyclins A and B1 measured in individual cells during their progression through G2 and mitosis.</a> Cytometry. 32, 71-77 (1998).</li><li>Langan, T. J., Chou, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synchronization+of+mammalian+cell+cultures+by+serum+deprivation.">Synchronization of mammalian cell cultures by serum deprivation.</a> Methods in Molecular Biology. 761, 75-83 (2011).</li><li>Kues, W. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cycle+synchronization+of+porcine+fetal+fibroblasts:+effects+of+serum+deprivation+and+reversible+cell+cycle+inhibitors.">Cell cycle synchronization of porcine fetal fibroblasts: effects of serum deprivation and reversible cell cycle inhibitors.</a> Biology of Reproduction. 62, 412-419 (2000).</li><li>Urbani, L., Sherwood, S. W., Schimke, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissociation+of+nuclear+and+cytoplasmic+cell+cycle+progression+by+drugs+employed+in+cell+synchronization.">Dissociation of nuclear and cytoplasmic cell cycle progression by drugs employed in cell synchronization.</a> Experimental Cell Research. 219, 159-168 (1995).</li></ol>

The authors have nothing to disclose.

La capacidad de analizar el ciclo celular es importante para la comprensión de la biología del cáncer y el mecanismo de acción de ambos fármacos y genes que influyen en la proliferación celular y el crecimiento. Mientras que hay una multitud de ensayos que miden la proliferación de células según se informa, la mayoría sólo proporciona una medida que indica las células número presente. Estos incluyen ensayos que miden el número de células por visualización directa y el recuento, la actividad metabólica o...

La evaluación de las características del ciclo celular y los cambios que se producen en las células durante la progresión del ciclo celular es fundamental para la comprensión de muchos aspectos de la biología, en particular la biología del cáncer. Muchos agentes en desarrollo para el tratamiento de tumores malignos tienen efectos profundos sobre la progresión del ciclo celular o inducir la muerte celular a través del ciclo celular dependiente de mecanismos. Con el fin de estudiar la dinámica del ciclo celular o células en una fase particular del ciclo celular, es habitual para sincronizar las células. Sin embargo métodos de sincronización pueden tener efectos perjudiciales sobre las células que se estudian, potencialmente confundiendo a los resultados obtenidos. 1 análisis Recientemente, el uso de proteínas etiquetadas con fluorescencia que sólo están presentes en fases particulares del ciclo de las células han permitido de la progresión del ciclo celular en las células individuales a través del tiempo 2, sin embargo las células a ser estudiadas necesidad de ser manipulados genéticamente para expresar estas proteínas etiquetadas, lo que limita su uso a sistemas en los que esto puede ser leidoslia logrado. El ciclo celular consiste de dos fases activas: la fase de síntesis (S), donde se replica el ADN y la mitosis (M), donde la división celular tiene lugar. Estas fases están separadas por tres fases gap, G 0, G 1 y G 2. G 0 o quiescencia, es una fase de reposo donde la célula ha dejado el ciclo, G 1 es donde las células aumentan de tamaño antes de la replicación del ADN y G 2, donde el crecimiento celular continúa entre la terminación de la replicación del ADN pero antes de la división celular. La progresión a través del ciclo celular está controlada por un número de puntos de control. El G 1 puesto de control se activa cuando las condiciones ambientales no son de apoyo de la síntesis de ADN e impide la entrada en fase S. El puesto de control de fase intra-S o el retraso pueden ser provocados por daños en el ADN que puede resultar en las horquillas de replicación estancado. Durante G 2 la fidelidad de la ADN replicado se confirma y si se detecta daño, entonces el G 2 </sub&gt; puesto de control se activa permitiendo la reparación del ADN antes de la división celular. Un punto de comprobación final durante la mitosis asegura que cromátidas se han alineado correctamente en la placa mitótico de modo que la división celular se puede completar con éxito. 3 La activación de estos puntos de control se utiliza comúnmente para sincronizar las poblaciones de células. Los puntos de control del ciclo celular pueden ser activados por un número de factores, pero en la biología del cáncer el más común es la detección de daños en el ADN. La respuesta al daño del ADN se inicia por la PI3-quinasa quinasas ataxia y telangiectasia Rad3 relacionados (ATR) y ataxia telangiectasia mutada (ATM) que activan las quinasas efectoras aguas abajo Chk1 y Chk2, respectivamente. 3 Una serie de eventos se activa Chk1 incluyendo estancado horquillas de replicación, entrecruzamientos de ADN, y el daño por radiación ultravioleta, mientras que Chk2 se activa principalmente por roturas de doble hebra. El método usual para estudiar el efecto de las condiciones alteradas de la longitud de la i ciclo celulars para sincronizar las células en una fase particular del ciclo celular. 1 Esto puede lograrse a través de varios métodos. Las células pueden separarse, físicamente basan en el tamaño, densidad, dispersión lateral (granularidad), y marcadores de expresión de la superficie celular. Más prácticamente, las células pueden ser sincronizados por medios químicos. Varios agentes tales como la timidina, la hidroxiurea y arabinósido de citosina se pueden utilizar para inhibir la síntesis de ADN en la fase S del ciclo celular que resulta en una acumulación de células en fase S que siguen ciclismo después se eliminan los agentes. Las células tratadas con nocodazol, que previene la formación del huso mitótico, arresto con un G 2 - o M-fase el contenido de ADN. Eliminación de suero a partir de los resultados de medio de cultivo en la acumulación de células en G 0 fase. El re-adición de los nutrientes dentro de las séricos cultura re-inicia el ciclo normal de las células. Sin embargo, todos estos métodos de sincronización interfieren con el ciclo normal y crecimiento de las células y puede Result en la muerte celular significativa. Sincronización de las células de la leucemia linfoblástica aguda es particularmente difícil y estas células no son susceptibles a la manipulación genética. El método aquí descrito permite la evaluación de la dinámica del ciclo celular y el estudio de células en fases particulares del ciclo celular sin sincronización tradicional o modificación genética. Este método también puede ser útil para otros tipos de células que no se consigue fácilmente la modificación genética y los procedimientos de sincronización tradicionales. El método se basa en el uso a largo establecido de bromodesoxiuridina (BrdU) incorporación, que tiene muy poco impacto en el crecimiento a corto plazo y la proliferación de las células. 4 protocolos establecidos BrdU toman ventaja de la incorporación de BrdU en el ADN recién sintetizado durante la fase S . Esto marca permanentemente las células como haber estado en la fase S durante la exposición BrdU. Esta población puede ser identificado en los puntos de tiempo posteriores por tinción para incor BrdUación y por lo tanto actúan como una población sincronizada que puede ser seguido y evaluado con el tiempo lo permite el estudio de los efectos del fármaco sobre el tránsito del ciclo celular. BrdU necesita ser expuesta antes de la tinción de anticuerpos, por lo general realizados a raíz de DNasa o tratamiento ácido 6,7. El uso de la citometría de flujo para detectar BrdU incorporado permite la inclusión de marcadores adicionales. El más importante es el uso de tintes para medir el contenido de ADN, lo que permite la evaluación de la distribución de fase del ciclo celular de las células que estaban en fase S en el inicio del estudio. 8 de superficie o intracelulares antígenos Además adicionales también pueden ser estudiados. 9 Estos pueden estar relacionados con los eventos del ciclo celular, como Ki67 o funciones celulares para aparentemente no relacionados, tales como marcadores de apoptosis como exfoliados caspasa-3. Las posibles aplicaciones están limitadas por la imaginación del 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 metodología se puede utilizar para obtener una gama de información. Algunas aplicaciones se describen aquí. Evaluación de la duración del ciclo celular Para determinar el tiempo requerido para que las células tránsito a través del ciclo celular, las células se cosecharon a diversos puntos de tiempo tras el pulso de BrdU. Los intervalos entre las evaluaciones pueden ser adaptadas a las células particulares que se analiza. Líneas celulares hematopoy...

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 describe el uso de bromodesoxiuridina (BrdU) para permitir la absorción de la seguimiento temporal de las células que estaban en fase S en un punto específico en el tiempo. La adición de colorantes de ADN y el etiquetado de anticuerpos facilita el análisis detallado del destino de las células en fase S en épocas posteriores.

Seguimiento temporal de la progresión del ciclo celular El uso de citometría de flujo sin la necesidad de sincronización

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

temporal-tracking-cell-cycle-progression-using-flow-cytometry-without

Research

Education

JoVE Journal

JoVE Core

Biology

Cellular Processes

Cell Cycle and Division

SCIENTISTS IN ACTION

19.0K vistas.  Westmead Millennium Institute for Medical Research and University of Sydney.  Este protocolo describe el uso de bromodesoxiuridina (BrdU) para permitir la absorción de la seguimiento temporal de las células que estaban en fase S en un punto específico en el tiempo. La adición de colorantes de ADN y el etiquetado de anticuerpos facilita el análisis detallado del destino de las células en fase S en épocas posteriores. 

Video: 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.

Citometría de flujo de purificación de las células de ratón 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 ...

Investigación

Biología

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.

Célula de medida Cinética la progresión del ciclo con el etiquetado metabólico y Citometría de Flujo

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 manchas y Proliferación métodos de modelización para la vigilancia del uso de tintes División Celular Celulares de seguimiento

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.

Celular seguimiento mediante fotoconvertible proteínas durante el desarrollo de pez cebra

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 para el Análisis de extracelular vesículas Usando citometría de flujo

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.

Sincronización de<em&gt; Caulobacter crescentus</em&gt; Para la Investigación del 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.

Thermo LED Flujo - Combinando optogenética con Citometría de Flujo

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.

Determinación de la superficie celular y Expresión relativa total de canales de iones recombinante mediante citometría de flujo

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.

Análisis de suspensiones celulares aisladas de tejidos sólidos por Spectral Citometría de Flujo

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.

Combina la sincronización celular mitótica y microscopia Confocal de alta resolución para estudiar el papel de las proteínas multifuncional ciclo celular durante la 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 ...