Este trabajo fue apoyado por el Premio a la Innovación Águilas (Fraternal Order of Eagles) y por la Fundación Mayo.

** Este protocolo de vídeo se basa en una publicación asociada 1: de alta sensibilidad de detección y análisis cuantitativo de los nativos interacciones proteína-proteína y complejos multiproteicos por citometría de flujo. Adam G. Schrum, Diana Gil, P. Elaine Dopfer, David L. Wiest, Laurence A. Turka, Wolfgang WA Schamel, Ed Palmer. STKE de la ciencia 2007 (389): PL2, 5 de junio de 2007, [DOI: 10.1126/stke.3892007pl2]. <a href="http://stke.sciencemag.org/cgi/content/abstract/2007/389/pl2">Por favor, haga clic aquí para ver esta publicación</a> . Antes de partir, Prepare las siguientes soluciones acuosas de archivo: <table><tbody><tr><td> MES acoplamiento Buffer: </td><td> Guárdela a 25 ° C </td></tr><tr><td> MES, pH 6,0 </td><td> 50 mM </td></tr><tr><td> EDTA </td><td> 1 mM </td></tr><tr><td> Enfriamiento / Bloqueo / Almacenamiento (SBC) Buffer: </td><td> Almacenar a 4 ° C </td></tr><tr><td> BSA </td><td> 1% </td></tr><tr><td> Azida de sodio </td><td> 0,02% </td></tr><tr><td> PBS, pH 7,4 </td><td> 1x </td></tr><tr><td> FCM Buffer: </td><td> Almacenar a 4 ° C </td></tr><tr><td> Tris, pH 7,4 </td><td> 50 mM </td></tr><tr><td> NaCl </td><td> 100 mM </td></tr><tr><td> Azida de sodio </td><td> 0,02% </td></tr><tr><td> FBS </td><td> 5% </td></tr><tr><td> PBS, pH 7,4 </td><td> 1x </td></tr></tbody></table> Preparar inmediatamente antes del uso: <table><tbody><tr><td> EDAC-MES: </td><td> Disolver 50 mg / ml polvo EDAC en tampón de acoplamiento MES </td></tr><tr><td> Digitonina solución (2% w / v): </td><td> Disuelva el polvo de digitonina en dH 2 O por calentamiento a 95 º C durante 5 minutos luego enfriar en hielo </td></tr><tr><td> Tampón de lisis: </td><td></td></tr><tr><td> Tris, pH 7,4 </td><td> 50 mM </td></tr><tr><td> NaCl </td><td> 150 mm </td></tr><tr><td> Digitonina solución </td><td> 1% </td></tr><tr><td> Inhibidores de la proteasa </td><td> 1x </td></tr><tr><td> Mantener en hielo </td><td></td></tr></tbody></table> 1. El acoplamiento de mAb a bolas <ol><li> Determinar la concentración de cuentas de las acciones compradas mediante la dilución en PBS y contar con un hemocitómetro. Comience con una dilución 1:10000 de cuentas para el conteo. </li><li> Pipeta de 18 x 10 6 granos en un tubo de microcentrífuga de 1,5 ml. </li><li> Lavar las perlas de 2 a 3 veces en 0,5 a 1,0 ml de tampón de acoplamiento MES, centrifugado a 20.000 g durante 3 minutos a 25 ° C después de cada lavado. </li><li> Volver a suspender las bolas en 50 l de acoplamiento Buffer MES. </li><li> Activar los grupos carboxilo de las cuentas mediante la adición de 20 l de recién preparada EDAC-MES. </li><li> Mezclar suavemente durante 15 minutos a 25 ° C de forma manual pipeteando arriba y abajo. </li><li> Lave las cuentas activadas 2 a 3 veces en 0,5 a 1,0 ml de PBS, centrifugar a 20.000 g durante 3 minutos a 25 ° C después de cada lavado. </li><li> Volver a suspender las cuentas activadas en 50 l de PBS. </li><li> Añadir 50 l de la IP MAB (0.2-1.0 en la concentración de las mg / mL) a la solución de cuentas activadas. </li><li> Mezcle por 3 a 4 horas a 25 º C colocando el tubo en un agitador vibratorio. Agite lo suficiente como para evitar la sedimentación de los granos en la parte inferior del tubo. </li><li> Lave mAb-junto cuentas 2 a 3 veces en 0,5 a 1,0 ml de PBS, centrifugar a 20.000 g durante 3 minutos a 25 ° C después de cada lavado. </li><li> Volver a suspender las bolas en 100 l de búfer SBC. Estos pueden ser almacenadas a 4 ° C. </li><li> La suspensión de las cuentas así, diluir 1:200-1:10,000 en PBS y contar con un hemocitómetro. La concentración debe ser medido para cada lote de preparación, por lo que el número de granos utilizados en cada experimento IP o pull-down puede ser controlada con precisión. </li></ol> 2. Post-nuclear lisado preparación e Ip El método de lisis y las condiciones óptimas de lisis dependerá del tipo de células y las interacciones entre proteínas que se examina. Una serie de protocolos existen y pueden ser modificados para su aplicación. <ol><li> Lisis de 20 x 10 6 &#39;pequeño&#39; células, como los linfocitos, o 5 x 10 6 &quot;grandes&quot; las células, como macrófagos o células tumorales, en 100 l buffer de lisis fresco en un tubo de microcentrífuga de 1,5 ml por 20 minutos en hielo. Escala el volumen de lisis, según sea necesario. </li><li> Para eliminar los núcleos y restos celulares insolubles, centrifugar el lisado a 20.000 g durante 10 min a 4 ° C. Mantenga el sobrenadante y descartar el pellet. </li><li> Añadir 0,5 x 10 5 a 2,5 x 10 5 mAb-junto a las cuentas de lisado clarificado, pipetear suavemente para mezclar. El volumen mínimo que utilizamos pararealizar una sola dirección IP es de 5 mL. </li><li> Colocar en una vertical de rotación de la rueda 4 horas para pasar la noche en una habitación fría. Ajuste a la velocidad suficiente para evitar que las cuentas de liquidación. Es aceptable que el líquido de bajo volumen de IPs para permanecer en el fondo del tubo durante la rotación. </li></ol> 3. Sondeo de bolas-capturado proteínas con anticuerpos conjugados a un fluorocromo <ol><li> Lave las cuentas IP dos veces en 0,2 a 1,0 ml de helado de buffer FCM, la centrifugación a 20.000 g a 4 ° C durante 3 min después de cada lavado. </li><li> Volver a suspender las bolas en 100 l de búfer y la FCM alícuota de 20 l en cada uno de cinco o más tubos de microcentrífuga de 1,5 mL o pocillos de una placa de chimenea de fondo. </li><li> Añadir fluorocromo conjugado AcMo a las muestras. Realizar la FCM mancha de acuerdo a las instrucciones del proveedor o de acuerdo a las concentraciones de determinados empíricamente. Como punto de partida, añadir 0,2 a 1 l de solución madre de anticuerpos (a 0.2-1.0 mg / mL) por tubo o pocillo e incubar durante 40 min en hielo. </li><li> Lave investigado cuentas en tubos de 1,5 ml dos veces en 1,0 ml de Buffer FCM helada, centrifugado a 20.000 g a 4 ° C durante 3 min después de cada lavado. Retire con cuidado el sobrenadante después de cada lavado, teniendo cuidado de no alterar las cuentas. Para una placa de chimenea de fondo, lavado dos veces con 0,2 ml de tampón helado FCM, centrifugar a 1000 g por 5 min a 4 ° C después de cada lavado. Una pipeta multicanal es útil para este paso. Para eliminar el sobrenadante de cuentas en una placa de chimenea de fondo, &#39;película&#39; de la placa una vez, entonces, al mismo tiempo al revés, borrar cualquier goteo de los bordes de los pozos. El volumen residual en cada pozo, que contiene las cuentas, será de unos 20 mL. </li><li> Volver a suspender las bolas en 200 Buffer FCM l por muestra. Traslado al etiquetado tubos FACS. Las muestras ya están listos para FCM. </li></ol> 4. FCM Adquisición Las cuentas de CML se describe en este protocolo son de 3 a 5 micras de diámetro, aproximadamente la mitad del diámetro de un linfocito del ratón en reposo. Por lo tanto, puede ser necesario aumentar manualmente la dispersión frontal (FSC) y la ganancia del amplificador dispersión lateral (SSC) de tensión a fin de que la población de los acontecimientos de cuentas para registrar en la escala. Individual cuentas IP deben formar una sola población cercanos entre sí. La configuración y la puerta debe ser ajustada para excluir a los dobletes de cuentas y los escombros. <ol><li> Antes de cuentas corrientes o las normas de cuentas, quitar la funda de contención de las gotas que rodea a la entrada de la muestra en algunos citómetros. Permita que el fluido de vaina a gotear entre las muestras, ya que se borrará la entrada y evitar cuentas de ser transferidos de una muestra a otra. </li><li> Utilice cuentas de etiqueta, un control negativo de la fluorescencia, y las partículas de arco iris de calibración (PCR), un control positivo de fluorescencia, para ajustar la configuración para que tanto los extremos positivos y negativos de fluorescencia aparecerá simultáneamente en el diario de escala del eje X. Esto asegura que la fluorescencia de las muestras experimentales también estará en gran escala. Tenga en cuenta que los PCR son más pequeñas que las cuentas de la LMC, por lo que el FSC y los parámetros de cooperación Sur-Sur, así como la posición de la puerta, puede ser necesario modificar temporalmente para esa muestra. </li><li> Volver a la etiqueta de perlas ajustes una vez calibrado con PCR se ha completado. Si sólo hay una sonda fluorescente por cada muestra se utiliza, sin compensación de fluorescencia es necesario. En general, la sonda complejos multiproteicos con un solo fluorocromo conjugado AcM por muestra FCM, y se examina la interacción de múltiples socios de tinción en paralelo muestras de cordón con mAbs específicas para las varias subunidades. </li><li> Adquirir y guardar archivos de datos de fluorescencia. Análisis puede llevarse a cabo utilizando el software de citometría de flujo, tales como CellQuest, FlowJo o cflow. </li></ol> 5. Resultados representante <img src="/files/ftp_upload/2066/2066fig1.jpg" alt="Figura 1" /> Figura 1. FCM-IP para el complejo multiproteico TCR/CD3. Células T de la cepa de ratones Balb / c fueron lisadas en el 1% digitonina, y el lisado fue sometido a IP-FCM con CD3ε anti-gotas. Los complejos capturados contenían cantidades significativas de TCR-β (región púrpura) y co-asociados CD3-ε (verde), pero, cerca de los niveles de fondo (que se define por la sonda inmunoglobulina irrelevante, rosa de seguimiento) de otras proteínas como Thy1.2 CD45, o H-2K (d) (marrón, naranja, azul y trazas, respectivamente). Ver resultados similares publicados previamente 1-6.

<ol>
	<li>Schrum, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-sensitivity+detection+and+quantitative+analysis+of+native+protein-protein+interactions+and+multiprotein+complexes+by+flow+cytometry.">High-sensitivity detection and quantitative analysis of native protein-protein interactions and multiprotein complexes by flow cytometry.</a> Sci STKE. , pl2-pl2 (2007).</li><li>Schrum, A. G. <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+multiprotein+complexes+by+flow+cytometry.">Visualization of multiprotein complexes by flow cytometry.</a> Curr Protoc Immunol. Chapter 5, 9-9 (2009).</li><li>Teixeiro, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+division+and+death+are+segregated+by+mutation+of+TCRbeta+chain+constant+domains.">T cell division and death are segregated by mutation of TCRbeta chain constant domains.</a> Immunity. 21, 515-526 (2004).</li><li>Teixeiro, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+T+Cell+receptor+signals+determine+CD8++memory+versus+effector+development.">Different T Cell receptor signals determine CD8+ memory versus effector development.</a> Science. 323, 502-505 (2009).</li><li>Treves, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Junctate+is+a+key+element+in+calcium+entry+induced+by+activation+of+InsP3+receptors+and/or+calcium+store+depletion.">Junctate is a key element in calcium entry induced by activation of InsP3 receptors and/or calcium store depletion.</a> J Cell Biol. 166, 537-548 (2004).</li><li>Gil, D., Schrum, A. G., Alarcon, B., Palmer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+receptor+engagement+by+peptide-MHC+ligands+induces+a+conformational+change+in+the+CD3+complex+of+thymocytes.">T cell receptor engagement by peptide-MHC ligands induces a conformational change in the CD3 complex of thymocytes.</a> J Exp Med. 201, 517-522 (2005).</li></ol>

Información acerca de las interacciones proteína-proteína es altamente relevante para el análisis de muchos procesos celulares como la transducción de señales, la maduración del linaje, la progresión del ciclo celular, apoptosis y cascadas. IP-FCM proporciona una forma rápida, cuantitativa y sensible para examinar la interacción de las proteínas y complejos de definir los miembros de multiproteico en su conformación nativa. Cuentas se pueden acoplar y se incubaron con lisados ​​de células en un día y se puede probar y analizar al día siguiente. Un formato de placa de 96 pocillos permite un gran número de muestras a analizar a la vez que ofrezcan servicios de recogida de datos eficiente para fines estadísticos o de tamizaje. Utilizando fluorescentes estándares de cuentas, el número de proteínas capturadas por cada cuenta puede ser estimado. Muy poco material de origen es necesaria para la captura y detección, lo que las muestras limitadas y analitos escasos todavía se pueden analizar las interacciones múltiples. A pesar de IP-FCM no requiere de la ingeniería genética, epítopo de etiquetado, desnaturalización, o in vitro, la mezcla de proteínas en un entorno no-fisiológico, se puede acoplar con estas y otras técnicas, por lo que es un instrumento valioso y accesible, con aplicabilidad a muchos sistemas biológicos. Solución de problemas: Muchos IP-FCM experimentos generar datos útiles de interacción de proteínas de la primera vez que se intentó. Sin embargo, la optimización de IP-FCM puede mejorar la conjugación de anticuerpos a los granos, la captura de proteínas complejas, y la sonda fluorescente vinculante. La eficiencia de la conjugación de anticuerpos IP puede ser determinado mediante el sondeo cuentas directamente acoplado con un anticuerpo anti-inmunoglobulina. Si esta eficiencia es baja, el aumento de la concentración de anticuerpos en la reacción de acoplamiento puede permitir que mayor cantidad de anticuerpos IP para conectar a cada cuenta. Esto puede aumentar la capacidad de unión de los lotes de cuentas IP, lo que resulta en la captura de mejora y la detección de analitos. Otros productos primarios-las moléculas que contienen aminas (por ejemplo, Tris, albúmina sérica bovina) no debe estar presente durante la reacción de acoplamiento, ya que pueden competir con el MAB para la fijación de cuentas y en consecuencia bajo acoplamiento mAb. Si la conjugación de mAb de cuentas resulta problemática por otras razones, en lugar de cuentas puede ser acoplado a avidina / estreptavidina, y posteriormente anticuerpos con biotina puede ser no-covalente y se utiliza para immunprecipitation. A pesar de una buena conjugación de anticuerpos, la detección inicial de cordón-fluorescencia asociada puede ser baja. La primera captura, el complejo en sí mismo puede ser baja. Debido a que IP-FCM depende de la concentración de los analitos, lo que aumenta el número de células lisadas por unidad de volumen de lisis puede aumentar la captura y detección de analitos. Además, la captura puede ser mejorada al reducir el número de cuentas de IP se incubaron con el lisado, que distribuye a través de complejos de captura menos granos, y los resultados de la fluorescencia mayor promedio por bolas al investigado. En segundo lugar, es posible que el acceso de los anticuerpos de la sonda a los complejos de captura es estéricamente impedido por el anticuerpo inmunoprecipitación. En este caso, el problema puede abordarse mediante el uso de diferentes anticuerpos para capturar y / o la sonda.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>carboxylate-modified polystyrene latex (CML) beads (5mm surfactant-free)</td>
<td>Reagent</td>
<td>Interfacial Dynamics Corporation/Molecular Probes</td>
<td>2-5000</td>
<td>Store at 4&deg;C.</td>
</tr>
<tr>
<td>Rainbow Calibration Particles</td>
<td>Reagent</td>
<td>Spherotech, Inc.</td>
<td>RCP-30-5A</td>
<td>Store at 4&deg;C.</td>
</tr>
<tr>
<td>2-[N-Morpholino] ethanesulfonic acid (MES)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>M-5287</td>
<td>&nbsp;</td>
<tr>
<td>1-Ethyl-3-(3-dimethylaminopropl) carbodiimide HCl (EDAC)</td>
<td>Reagent</td>
<td>Pierce Sigma</td>
<td>22980 E-6383</td>
<td>Store at -20&deg;C under desiccating conditions.</td>
</tr>
<tr>
<td>Protease Inhibitor General Use Cocktail, containing AEBSF, Bestatin, Aprotinin, EDTA, E-64, and Leupeptin</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P-2714</td>
<td> Prepare 100x stock solution in H2O. Aliquot and store at -20&deg;C. Handle with gloves.</td>
</tr>
<tr>
<td>PCR plates, 96-well 0.2mL, natural, tall chimney</td>
<td>Reagent</td>
<td>Fisher</td>
<td>08-408-230</td>
<td>For use in place of microcentrifuge tubes during FCM staining</td>
</tr>		</tbody>
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ip-fcm:  immunoprecipitation detected by flow cytometry

Inmunoprecipitación detectada mediante citometría de flujo (IP-FCM) es un método eficaz para la detección y cuantificación de interacciones proteína-proteína. El principio básico es extensivo de ELISA sandwich, en la cual puede ser el analito capturado primaria detectados junto con otras moléculas físicamente asociados dentro de los complejos multiproteico. El procedimiento consiste en el acoplamiento covalente de microesferas de látex de poliestireno con inmunoprecipitación anticuerpos monoclonales (mAb) específico para una proteína de interés, la incubación de estas cuentas con lisados ​​de células, el sondeo capturado complejos de proteínas con el conjugado a un fluorocromo sondas, y el análisis de cuentas asociadas a fluorescencia por citometría de flujo. IP-FCM es extremadamente sensible, permite el análisis de proteínas en su país natal (no desnaturalizado) del estado, y se presta a ninguno de los análisis semi-cuantitativo o cuantitativo. Como ventajas adicionales, IP-FCM no requiere de la ingeniería genética o equipo especializado, que no sea un citómetro de flujo, y puede ser fácilmente adaptada para aplicaciones de alto rendimiento.

Watch this Scientific Journal Video about IP-FCM:  Immunoprecipitation Detected by Flow Cytometry at JoVE.com

IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

El método IP-FCM se presenta, lo que permite un sensible, sólida evaluación, bioquímicos nativos de interacciones proteína-proteína, sin necesidad de la ingeniería genética o muestras de gran tamaño.

IP-FCM: inmunoprecipitación detectada por citometría de flujo

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic ...

ip-fcm-immunoprecipitation-detected-by-flow-cytometry

Research

JoVE Journal

Biology

17.4K vistas.  Mayo Clinic. El método IP-FCM se presenta, lo que permite un sensible, sólida evaluación, bioquímicos nativos de interacciones proteína-proteína, sin necesidad de la ingeniería genética o muestras de gran tamaño.

Video: IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes.
The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body1. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes2,3.
The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies.
Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.

This protocol describes a rapid technique to quantify the translocation of GLUT4 from the cytoplasm to the plasma membrane of cells by flow cytometry.

La medición cuantitativa de la translocación de GLUT4 a la membrana plasmática por citometría de flujo

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

Investigación

Biología

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 ...

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 ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

Las mediciones del calcio citosólico en las células epiteliales renales mediante citometría de flujo

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and pathophysiology. This protocol describes a technique where mitochondria isolated from rodent tissue are immunolabeled and analyzed by flow cytometry. Mitochondria are isolated from rodent spinal cords and subjected to a rapid enrichment step so as to remove myelin, a major contaminant of mitochondrial fractions prepared from nervous tissue. Isolated mitochondria are then labeled with an antibody of choice and a fluorescently conjugated secondary antibody. Analysis by flow cytometry verifies the relative purity of mitochondrial preparations by staining with a mitochondrial specific dye, followed by detection and quantification of immunolabeled protein. This technique is rapid, quantifiable and high-throughput, allowing for the analysis of hundreds of thousands of mitochondria per sample. It is applicable to assess novel proteins at the mitochondrial surface under normal physiological conditions as well as the proteins that may become mislocalized to this organelle during pathology. Importantly, this method can be coupled to fluorescent indicator dyes to report on certain activities of mitochondrial subpopulations and is feasible for mitochondria from the central nervous system (brain and spinal cord) as well as liver.

Described here is a method to detect and quantify mitochondrial outer membrane proteins by immunolabeling of mitochondria isolated from rodent tissue and analysis by flow cytometry. This method can be extended to assess functional aspects of mitochondrial subpopulations.

La inmunodetección de la membrana externa proteínas por Citometría de Flujo de mitocondrias aisladas

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and ...

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle &#8220;in a dish&#8221; for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.

The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population.

Alta Eficiencia Diferenciación de células madre pluripotentes humanas a cardiomiocitos y Caracterización por citometría de flujo

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the ...

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.

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 ...

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 ...

Measuring Endoreduplication by Flow Cytometry of Isolated Tuber Protoplasts

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated with specialization, development and increase in cellular size. In plants, endoreduplication seems to facilitate the growth and expansion of certain tissues and organs. Among them is the tuber of potato (Solanum tuberosum), which undergoes considerable cellular expansion in fulfilling its function of carbohydrate storage. Thus, endoreduplication may play an important role in how tubers are able to accommodate this abundance of carbon. However, the cellular debris resulting from crude nuclear isolation methods of tubers, methods that can be used effectively with leaves, precludes the estimation of the tuber endoreduplication index (EI). This article presents a technique for assessing tuber endoreduplication through the isolation of protoplasts while demonstrating representative results obtained from different genotypes and compartmentalized tuber tissues. The major limitations of the protocol are the time and reagent costs required for sample preparation as well as relatively short lifespan of samples after lysis of protoplasts. While the protocol is sensitive to technical variation, it represents an improvement over traditional methods of nuclear isolation from these large specialized cells. Possibilities for improvements to the protocol such as recycling enzyme, the use of fixatives, and other alterations are proposed.

The protocol described herein is a method for measuring endoreduplication within tubers of potato (Solanum tuberosum). It includes plasmolysis and protoplast extraction steps to decrease the noise and debris in downstream flow cytometric analysis.

Endorreduplicación medición por citometría de flujo de protoplastos aislados de tubérculos

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated ...

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion and the spread of neurodegenerative diseases. The commonly used laboratory strains of the amoeba Dictyostelium discoideum have extremely high fluid uptake rates when grown in nutrient medium, over 90% of which is due to macropinocytosis. In addition, many of the known core components of mammalian macropinocytosis are also present, making it an excellent model system for studying macropinocytosis. Here, the standard technique to measure internalized fluid using fluorescent dextran as a label is adapted to a 96-well plate format, with the samples analyzed by flow cytometry using a high-throughput sampling (HTS) attachment.
Cells are fed non-quenchable fluorescent dextran for a pre-determined length of time, washed by immersion in ice-cold buffer and detached using 5 mM sodium azide, which also stops exocytosis. Cells in each well are then analyzed by flow cytometry. The method can also be adapted to measure membrane uptake and phagocytosis of fluorescent beads or bacteria.
This method was designed to allow measurement of fluid uptake by Dictyostelium in a high-throughput, labor and resource efficient manner. It allows simultaneous comparison of multiple strains (e.g. knockout mutants of a gene) and conditions (e.g. cells in different media or treated with different concentrations of inhibitor) in parallel and simplifies time-courses.

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Macropinocytosis, large-scale non-specific fluid uptake, is important in many areas of clinical biology including immunology, infection, cancer, and neurodegenerative diseases. Here, existing techniques have been adapted to allow high-throughput, single-cell resolution measurement of macropinocytosis in the macropinocytosis model organism Dictyostelium discoideum using flow cytometry.

Medida de alto rendimiento de macropinocitosis discoideum de Dictyostelium por citometría de flujo

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion ...