Este proyecto fue apoyado en parte por los dineros del Estado de Florida de Scripps y números premio R01GM085079 R21HD061304 y del Instituto Nacional de Ciencias Médicas Generales y el Instituto Nacional de Salud Infantil y Desarrollo Humano, respectivamente. Este es el número 20917 manuscrito de The Scripps Research Institute.

Este protocolo se puede separar en dos etapas principales: (1) la disociación y Hoechst 33342 tinción de células de los testículos de ratón seguido, si es necesario, (2) FACS clasificación de las fracciones correspondientes meiótica, incluyendo todas las etapas de la meiosis, las células germinales de espermátidas redondas. Una vez recogidos, estas poblaciones altamente enriquecido meiótica puede ser utilizado para una amplia gama de análisis. Este protocolo describe la disociación de un testículo adulto, los volúmenes se pueden adaptar en consecuencia de los menores o de los testículos adicionales. 1. La disociación testículos <ol><li> Coloque un testículo decapsulated en un tubo de 15 ml. </li><li> Añadir 3 ml de solución salina equilibrada de Gey (GBSS), que contiene 120 U / ml de colagenasa tipo I. </li><li> Añadir 10 l de DNasa I (1mg/ml solución de valores en el 50% de glicerol) y agitar enérgicamente a mano hasta que vea los túbulos testiculares comienzan a disociarse. </li><li> Agitar horizontalmente a un máximo de 120 rpm durante 15 minutos a 33 ° C. </li><li> Se decanta por 1 min vertical a temperatura ambiente y desechar el sobrenadante. </li><li> Repita los pasos 1,2 a 1,5. </li><li> Añadir 2,5 ml de GBSS contiene colagenasa tipo I, 50 l de una solución de reserva 50mg/ml tripsina resuspendido en solución de HCl 1 mM, y 10 l de DNasa I (1mg/ml), e invertir el tubo varias veces. </li><li> Agitar horizontalmente a un máximo de 120 rpm durante 15 minutos a 33 ° C. </li><li> Utilizando una pipeta Pasteur de plástico desechables, con orificio de ancho, pipeta suavemente hacia arriba y hacia abajo por 3 grupos min.No debe ser visible en este momento. </li><li> Añadir 30 l de tripsina, 10 l de DNAsa I, 40 l de Hoechst 33342 resuspendieron en DMSO (10 mg / ml), e invertir el tubo varias veces. </li><li> Agitar horizontalmente a un máximo de 120 rpm durante 15 minutos a 33 ° C. </li><li> Añadir 400 l de suero fetal bovino (FCS) y mezclar invirtiendo para inactivar la tripsina. </li><li> Tinción final se realiza mediante la adición de 50 l de Hoechst 33342 resuspendieron en DMSO (10 mg / ml), y 10 l de DNasa I (1 mg / ml). </li><li> Agitar horizontalmente a un máximo de 120 rpm durante 15 minutos a 33 ° C. </li><li> La muestra de testículo disociada se pasa a través de dos GBSS de 40 micras previamente mojado filtros desechables en un tubo cónico de 50 ml, 5 l de yoduro de propidio (PI) y la solución se añade la muestra se mezcla suavemente con la pipeta varias veces con desechables Pasteur pipeta. </li><li> Muestra es transferida a una jeringa de plástico de 5 ml a través de una aguja de calibre 18. Este último es sustituido por una aguja de calibre 22 para la entrega de la muestra. La jeringa se almacena en hielo y protegido de la luz hasta que esté listo para el procesamiento de FACS. </li></ol> 2. FACS configuración y purificación <ol><li> Clasificación se realizó en un clasificador de Becton-Dickinson Aria celular IIU. Las condiciones descritas por lo tanto, se debe utilizar como punto de partida, especialmente cuando se utilizan equipos diferentes. Hemos utilizado las condiciones anteriormente descritas 7,8. </li><li> Dado que la IIU Aria no tiene un láser UV, adaptamos el protocolo para la detección de manchas Hoescht uso de un láser violeta de 405 nm, además de que utiliza un láser azul 488 nm para la detección de dispersión frontal y lateral. Además, algunos filtros se modificaron con el fin de limitar algunos filtración de láser rojo que resulta en la nitidez mejorada trama dispersa. </li><li> El láser violeta se ha configurado con un pase de banda 450/40nm para la detección de Hoescht azul de emisión y un pase de banda 585/42nm de Hoescht emisión roja. Un pase largo 502nm se utiliza para separar azul de fluorescencia de color rojo. </li><li> Una boquilla de 100 micras se utilizan con una frecuencia de caída de la unidad de 28.000 gotas / segundo. La tasa de umbral de la muestra fue de aproximadamente 4.000 eventos / segundo. La opción de control de la temperatura se utiliza para mantener la muestra y los tubos de recogida a 4 ° C toda la duración de la clasificación. Además, la función de la agitación de la muestra se utilizó a 200 rpm para evitar que la muestra de la sedimentación a lo largo de la clase. </li><li> Por lo general, dos o tres testículos fueron procesados ​​por 6 horas clase que permite la obtención de 0,5-2.0x10 6 células de cada población (véase la sección representativa). </li><li> La muestra fue ordenada en partes alícuotas de aproximadamente 750μl dispensado desde la jeringa. Mientras tanto, durante estas pausas los tubos de recolección se mantuvieron a 4 ° C, protegido de la luz, y se mezclan suavemente antes de ordenar la reanudación. </li><li> Las muestras fueron ordenados en un tubo de recogida de vidrio borosilicatado 12x75mm contiene 250μl DMEM suplementado con 10% FCS. </li></ol> 3. Los resultados representativos: Un típico perfil de FACS se muestra en la Figura 1. Todos los pasos más importantes de la meiosis se identifican y se indican en la leyenda. Si el Hoechst 33342 tinción no es óptimo, el perfil global parece mucho más compacto y menos definida. Un problema común se refiere a que no agrega suficiente DNAsa, lo que induce aglutinación rápida de las células. Además de DNAsa adicional en todos los pasos procesados ​​por lo general resuelve este problema. Congelar ªintimidar las acciones de DNAsa (almacenadas a -20 ° C) debe ser reducido al mínimo, ya que esto da lugar a una rápida pérdida de actividad de la enzima. Spo11 es la endonucleasa meiótica que dirige la doble cadena se rompe en los sitios de sitios de recombinación meiótica 12. La figura 2 muestra los perfiles típicos de un testículo Spo11 nulo en doble capítulo romper no se forman, lo que resulta en una meiosis abortiva 12, así como una pre-puberous perfil de un ratón de 13 días de edad, lo que revela la naturaleza asíncrona de esta primera ola de la meiosis. Estándar de análisis citogenético mediante la combinación de proteínas complejas sinaptonémico 3 (SCP3) y fosforilados anticuerpos histona H2AX etapa de marcadores específicos de la meiosis debe ser utilizado inicialmente para confirmar la pureza y la naturaleza de las células meióticas ordenados, como se describe elswhere 11. <img src="/files/ftp_upload/2602/2602fig1.jpg" alt="Figura 1" /> Figura 1. Tipo salvaje FACS meiosis perfiles. A) diagrama de dispersión se muestra Representante. Las células cerradas se muestran. Tenga en cuenta la gran cantidad de residuos presentes en los testículos adultos que contienen principalmente las membranas vacías y las colas de espermatozoides. B) Un representante parcela PI muestra la cantidad muy limitada de células PI positivas presentes en una muestra. La puerta típica se muestra. C) Un representante de tipo salvaje Hoechst 33342 se muestra el perfil de FACS. Las distintas poblaciones de células meióticas que puede ser purificado mediante este método para el estudio adicional se indican: espermatogonias (E), pre-leptotene (PL), leptotene-zygotene (L / Z), a principios de Paquiteno (PE), el medio Paquiteno ( MP), de finales del Paquiteno (LP), diplotene (D), y espermátidas redondas (RS). <img src="/files/ftp_upload/2602/2602fig2.jpg" alt="Figura 2" /> Figura 2. Spo11 nulo y de tipo salvaje día 13 perfiles FACS meióticas. A) Una típica Spo11 nulo perfil se muestra con un claro fracaso meiótica, donde puede haber más allá de la etapa leptotene / zygotene temprana pachytene detectado. B) El perfil de una de 13 días de edad, de tipo salvaje ratones machos se muestra la alta concentración de células leptotene / zygotene. Esta primera oleada de la meiosis es bastante asíncrono, como claramente se visualizan mediante esta metodología.

<ol>
	<li>Meistrich, M. L., Bruce, W. R., Clermont, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+composition+of+fractions+of+mouse+testis+cells+following+velocity+sedimentation+separation.">Cellular composition of fractions of mouse testis cells following velocity sedimentation separation.</a> Exp. Cell Res. 79, 213-227 (1973).</li><li>Grabske, R. J., Lake, S., Gledhill, B. L., Meistrich, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrifugal+elutriation:+separation+of+spermatogenic+cells+on+the+basis+of+sedimentation+velocity.">Centrifugal elutriation: separation of spermatogenic cells on the basis of sedimentation velocity.</a> J. Cell. Physiol. 86, 177-189 (1975).</li><li>Purification, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=culture,+and+fractionation+of+spermatogenic+cells.">culture, and fractionation of spermatogenic cells.</a> Methods Enzymol. 225, 84-113 (1993).</li><li>Romrell, L. J., Bellve, A. R., Fawcett, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+mouse+spermatogenic+cells+by+sedimentation+velocity.+A+morphological+characterization.">Separation of mouse spermatogenic cells by sedimentation velocity. A morphological characterization.</a> Dev. Biol. 49, 119-131 (1976).</li><li>Meistrich, M. L., Trostle, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+mouse+testis+cells+by+equilibrium+density+centrifugation+in+renografin+gradients.">Separation of mouse testis cells by equilibrium density centrifugation in renografin gradients.</a> Exp. Cell Res. 92, 231-244 (1975).</li><li>Namekawa, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postmeiotic+sex+chromatin+in+the+male+germline+of+mice.">Postmeiotic sex chromatin in the male germline of mice.</a> Curr. Biol. 16, 660-667 (2006).</li><li>Lassalle, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Side+Population'+cells+in+adult+mouse+testis+express+Bcrp1+gene+and+are+enriched+in+spermatogonia+and+germinal+stem+cells.">Side Population' cells in adult mouse testis express Bcrp1 gene and are enriched in spermatogonia and germinal stem cells.</a> Development. 131, 479-487 (2004).</li><li>Bastos, H. <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+characterization+of+viable+meiotic+and+postmeiotic+cells+by+Hoechst+33342+in+mouse+spermatogenesis.">Flow cytometric characterization of viable meiotic and postmeiotic cells by Hoechst 33342 in mouse spermatogenesis.</a> Cytometry A. 65, 40-49 (2005).</li><li>Chowdhury, R., Bois, P. R., Feingold, E., Sherman, S. L., Cheung, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+analysis+of+variation+in+human+meiotic+recombination.">Genetic analysis of variation in human meiotic recombination.</a> PLoS Genet. 5, e1000648-e1000648 (2009).</li><li>Roig, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+TRIP13/PCH2+is+required+for+recombination+and+normal+higher-order+chromosome+structure+during+meiosis.">Mouse TRIP13/PCH2 is required for recombination and normal higher-order chromosome structure during meiosis.</a> PLoS Genet. 6, (2010).</li><li>Getun, I. V., Wu, Z. K., Khalil, A. M., Bois, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleosome+occupancy+landscape+and+dynamics+at+mouse+recombination+hotspots.">Nucleosome occupancy landscape and dynamics at mouse recombination hotspots.</a> EMBO Rep. 11, 555-560 (2010).</li><li>Romanienko, P. J., Camerini-Otero, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+Spo11+gene+is+required+for+meiotic+chromosome+synapsis.">The mouse Spo11 gene is required for meiotic chromosome synapsis.</a> Mol. Cell. 6, 975-987 (2000).</li></ol>

El protocolo presentado en este documento permite purificar al mismo tiempo de ratones machos adultos de toda la gama de las células del estadio meiótico con una pureza muy alta, permitiendo a los investigadores estudiar la dinámica de este proceso fundamental. Células purificadas se pueden utilizar para numerosas aplicaciones que van desde la extracción de RNA 9,10, la cartografía nucleosoma 11, la detección de moléculas recombinantes, análisis de proteína, y muchos más. Sin embargo, lo...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Collagenase Type-1</td>
<td>&nbsp;</td>
<td>Worthington</td>
<td>CLSS1</td>
<td>Dissolve to 12,000U/ml in GBSS, store at 4&deg;C</td>
</tr>
<tr>
<td>Trypsin</td>
<td>&nbsp;</td>
<td>Worthington</td>
<td>TRL3</td>
<td>Dissolve in 1mM HCl to 50mg/ml, store at 4&deg;C</td>
</tr>
<tr>
<td>Hoechst 33342</td>
<td>&nbsp;</td>
<td>Arcos</td>
<td>230001000</td>
<td>Saturated solution (10mg/ml in DMSO, store at 4&deg;C</td>
</tr>
<tr>
<td>DNAse I</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>DNEP</td>
<td>Dissolve in water/50% glycerol to 1mg/ml, store at -20&deg;C</td>
</tr>
<tr>
<td>Gey&#x2019;s Balance Salt Solution </td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>G9779</td>
<td>&nbsp;</td>
<tr>
<td>6" Transfer Pipet</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>137119D</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

flow cytometry purification of mouse meiotic cells

La naturaleza heterogénea de tipos de células en los testículos y la ausencia de modelos de cultivo celular meiótica se han obstáculos importantes para el estudio de los programas de diferenciación única que se manifiestan durante la meiosis. Dos métodos principales se han desarrollado para purificar, en distintos grados, distintas fracciones meiótica de adultos y animales inmaduros: elutriación o Staput (sedimentación) utilizando BSA y / o gradientes de Percoll. Ambos métodos se basan en el tamaño celular y la densidad para separar las células meióticas 1-5. En general, a excepción de las poblaciones de células pocos 6, estos protocolos no dar suficiente pureza de las poblaciones de células meióticas numerosos que son necesarios para los análisis moleculares detallados. Además, con estos métodos por lo general un tipo de células meióticas puede ser purificado en un momento dado, lo que añade un nivel adicional de complejidad en relación a la reproducibilidad y la homogeneidad en la comparación de muestras de células meióticas. Aquí se describe un método perfeccionado que permite visualizar fácilmente, identificar y purificar las células meióticas, a partir de células germinales de espermátidas redondas, usando FACS combinado con Hoechst 33342 tinción 7,8. Este método proporciona una visión global del proceso meiótico completo y permite purificar altamente células viables de la mayoría de la etapa de la meiosis. Estas células purificadas se pueden analizar en detalle los cambios moleculares que acompañan a la progresión a través de la meiosis, por ejemplo, cambios en la expresión génica 9,10 y la dinámica de ocupación nucleosoma en los hotspots de la recombinación meiótica 11.

Watch this Scientific Journal Video about Flow Cytometry Purification of Mouse Meiotic Cells at JoVE.com

Flow Cytometry Purification of Mouse Meiotic Cells

Un método eficaz para obtener fracciones altamente purificadas meiótica viables de ratón testículos se describe, que combina un refinado protocolo de disociación celular, con la clasificación de células activadas fluorescentes (FACS). Este método aprovecha las diferencias en el contenido de ADN nuclear y la densidad de las fracciones discretas meióticas.

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 ...

flow-cytometry-purification-of-mouse-meiotic-cells

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Biology

Unit 1

Meiosis

SCIENTISTS IN ACTION

17.5K vistas.  The Scripps Research Institute. Un método eficaz para obtener fracciones altamente purificadas meiótica viables de ratón testículos se describe, que combina un refinado protocolo de disociación celular, con la clasificación de células activadas fluorescentes (FACS). Este método aprovecha las diferencias en el contenido de ADN nuclear y la densidad de las fracciones discretas meióticas.

Video: Flow Cytometry Purification of Mouse Meiotic Cells

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

Tinción de las proteínas en geles con Coomassie G-250, sin ácido disolvente orgánico y el acético

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Investigación

Biología

Flow Cytometry-based Purification of S. cerevisiae Zygotes

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae zygotes result from the fusion of haploid cells of distinct mating type (MATa, MATalpha) and give rise to corresponding stable diploids that successively generate as many as 20 diploid progeny as a result of their strikingly asymmetric mitotic divisions 2. Zygote formation is orchestrated by a complex sequence of events: In this process, soluble mating factors bind to cognate receptors, triggering receptor-mediated signaling cascades that facilitate interruption of the cell cycle and culminate in cell-cell fusion. Zygotes may be considered a model for progenitor or stem cell function.
Although much has been learned about the formation of zygotes and although zygotes have been used to investigate cell-molecular questions of general significance, almost all studies have made use of mating mixtures in which zygotes are intermixed with a majority population of haploid cells 3-8. Many aspects of the biochemistry of zygote formation and the continuing life of the zygote therefore remain uninvestigated.
Reports of purification of yeast zygotes describe protocols based on their sedimentation properties 9; however, this sedimentation-based procedure did not yield nearly 90% purity in our hands. Moreover, it has the disadvantage that cells are exposed to hypertonic sorbitol. We therefore have developed a versatile purification procedure. For this purpose, pairs of haploid cells expressing red or green fluorescent proteins were co-incubated to allow zygote formation, harvested at various times, and the resulting zygotes were purified using a flow cytometry-based sorting protocol. This technique provides a convenient visual assessment of purity and maturation. The average purity of the fraction is approximately 90%. According to the timing of harvest, zygotes of varying degrees of maturity can be recovered. The purified samples provide a convenient point of departure for "-omic" studies, for recovery of initial progeny, and for systematic investigation of this progenitor cell.

Flow Cytometry-based Purification of S. cerevisiae Zygotes

To purify zygotes of S. cerevisiae, haploid cells of opposite mating type were engineered to express red or green fluorescent proteins, co-incubated to allow zygote formation, and fractionated using a flow cytometry-based protocol. The highly-enriched fraction enables subsequent "-omic" studies, recovery of initial progeny, and systematic investigation of zygote morphogenesis.

Citometría de flujo basado Purificación de<em&gt; S. cerevisiae</em&gt; Los cigotos

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae ...

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 ...

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 ...

Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on the surface of slides (rather than dropping them from a height), we demonstrate an optimized technique on mouse spermatocytes that was first described in 1997. This method is widely used in laboratories to study mammalian meiosis because it yields a plethora of high quality nuclei undergoing substages of prophase I. Seminiferous tubules are first placed in a hypotonic solution to swell spermatocytes. Then spermatocytes are released into a sucrose solution to create a cell suspension, and nuclei are spread onto fixative-soaked glass slides. Following immunostaining, a diversity of proteins germane to meiotic processes can be examined. For example, proteins of the synaptonemal complex, a tripartite structure that connects the chromosome axes/cores of homologs together can be easily visualized. Meiotic recombination proteins, which are involved in repair of DNA double-strand breaks by homologous recombination, can also be immunostained to evaluate progression of prophase I. Here we describe and demonstrate in detail a technique widely used to study mammalian meiosis in spermatocytes from juvenile or adult male mice.

Meiosis is the developmental process by which gametes are formed through a single round of DNA replication and two successive rounds of chromosome segregation. Mammalian meiosis can be examined by utilizing a technique to prepare meiotic chromosome spreads. Here, we demonstrate a method of preparing surface-spread nuclei from mouse spermatocytes.

Preparación del cromosoma meiótico se extiende desde ratón espermatocitos

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on ...

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 ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

Trans-interno celular masiva inyección de células madre embrionarias conduce a mayores tasas de quimerismo

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Isolation and Characterization of the Immune Cells from Micro-dissected Mouse Choroid Plexuses

The brain is no longer considered as an organ functioning in isolation; accumulating evidence suggests that changes in the peripheral immune system can indirectly shape brain function. At the interface between the brain and the systemic circulation, the choroid plexuses (CP), which constitute the blood-cerebrospinal fluid barrier, have been highlighted as a key site of periphery-to-brain communication. CP produce the cerebrospinal fluid, neurotrophic factors, and signaling molecules that can shape brain homeostasis. CP are also an active immunological niche. In contrast to the brain parenchyma, which is populated mainly by microglia under physiological conditions, the heterogeneity of CP immune cells recapitulates the diversity found in other peripheral organs. The CP immune cell diversity and activity change with aging, stress, and disease and modulate the activity of the CP epithelium, thereby indirectly shaping brain function. The goal of this protocol is to isolate murine CP and identify about 90% of the main immune subsets that populate them. This method is a tool to characterize CP immune cells and understand their function in orchestrating periphery-to-brain communication. The proposed protocol may help decipher how CP immune cells indirectly modulate brain function in health and across various disease conditions.

This study uses flow cytometry and two different gating strategies on isolated perfused mice brain choroid plexuses; this protocol identifies the main immune cell subsets that populate this brain structure.

Aislamiento y caracterización de las células inmunes de plexos coroideos de ratón microdisecados

The brain is no longer considered as an organ functioning in isolation; accumulating evidence suggests that changes in the peripheral immune system can ...

Isolation and Identification of Vascular Endothelial Cells from Distinct Adipose Depots for Downstream Applications

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone regulation, barrier functions, and angiogenesis. Endothelial cell dysfunction is a hallmark predictor and major driver for the progression of severe cardiovascular diseases, yet the underlying mechanisms remain poorly understood. The ability to isolate and perform analyses on endothelial cells from various vascular beds in their native form will give insight into the processes of cardiovascular disease. This protocol presents the procedure for the dissection of mouse subcutaneous and mesenteric adipose tissues, followed by isolation of their respective arterial vasculature. The isolated arteries are then digested using a specific cocktail of digestive enzymes focused on liberating functionally viable endothelial cells. The digested tissue is assessed by flow cytometry analysis using CD31+/CD45&#8722; cells as markers for positive endothelial cell identification. Cells can be sorted for immediate downstream functional assays or used to generate primary cell lines. The technique of isolating and digesting arteries from different vascular beds will provide options for researchers to evaluate freshly isolated vascular cells&#160;from arteries of interest and allow them to perform a wide range of functional tests on specific cell types.

This protocol details a method for the dissection of mouse adipose depots and the isolation and digestion of respective arteries to liberate and then identify the endothelial cell population. Freshly isolated cells used in downstream applications will advance the understanding of vascular cell biology and the mechanisms of vascular dysfunction.

Aislamiento e identificación de células endoteliales vasculares de distintos depósitos adiposos para aplicaciones posteriores

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone ...