Los autores desean agradecer al Dr. Leonid Volkov y Éric Bouchard por su asesoramiento técnico en relación con la microscopía de epifluorescencia. Apoyo financiero Financiado por los Institutos Canadienses de Investigación en Salud (subvención # MOP-93781) de GB

Cuidado de los animales estaba en conformidad con el cuidado de los animales y el uso comité de la Universidad de Sherbrooke. 1. Preparación del tubo <ol><li> El día antes de la clasificación de células, añadir 1-2 ml de suero bovino fetal inactivado por calor (FBS) a 5 ml de polipropileno tubos de fondo redondo, y a 15 ml y 50 ml tubos cónicos de polipropileno. Paso crítico: Asegúrese de que todos los tubos utilizados en el protocolo es revestido. Nota: recubrimiento FBS evita que las células germinales se pegue a las paredes del tubo. </li><li> Poco a poco la mezcla de O / N por inversión a 4 ° C para revestir usando los tubos de manera uniforme un rotador. </li><li> El día siguiente, eliminar FBS a partir de tubos recubiertos por decantación. <ol><li> * Paso crítico: Asegúrese de que los 5 ml de polipropileno tubos de fondo redondo y los 15 y 50 ml tubos utilizados para la preparación de células (protocolo de los pasos 2.1 a 2.11) están completamente vacíos de FBS para evitar Fanning (es decir, la desviación imprecisa de las corrientes laterales). Utilice una pipeta P200 para eliminar las FBS. <br/&gt; Nota: Residual FBS en los tubos antes de la clasificación puede conducir a una pérdida de las células o la contaminación de otros tubos utilizados durante la clasificación de células, </li><li> Deja un pequeño volumen residual de FBS (alrededor de 100 a 200 l) en el 5 ml de polipropileno ronda tubos de fondo utilizados para la recolección de células purificadas. </li></ol></li></ol> 2. Preparación de la célula <ol><li> Sacrificar uno ratón macho bajo anestesia usando O 2 / isoflurano (inducción al 5% después se mantuvo a 2%), seguido por asfixia con CO2. </li><li> Impuestos Especiales y desencapsulan ambos testículos y picar con pequeñas tijeras en 500 l de tampón de clasificación en un tubo de 1,5 ml. </li><li> Células germinales rasantes de túbulos seminíferos de suave pipeteo hacia arriba y abajo en el tubo de 1,5 ml, primero con un tronco de 100-1.000 l punta, y luego con una punta de 100-1.000 l intacta. </li><li> Retirar los escombros y matas por el paso uno de filtración usando un filtro de células de 40 micras y recoger filtrado en los 50 ml prev tubo cónicoiamente recubierto con FBS en el paso 1. </li><li> Lavar el filtro una vez con tampón de clasificación hasta un volumen total de 3 ml y la transferencia de filtrado en el tubo cónico de 15 ml previamente recubierto en el paso 1. </li><li> Añadir 16 l de EDTA 50 mM pH 8 por mililitro de suspensión celular (48 l para 3 ml) para obtener una concentración final de EDTA 0,8 mM. </li><li> Para fijar las células germinales, se añade lentamente 3 volúmenes de etanol al 100% enfriado con hielo utilizando una pipeta de 10 ml con agitación suave (vórtice a baja velocidad), que producirá una suspensión lechosa. Paso crítico: añadiendo lentamente etanol es crucial e importante para preservar la integridad de la preparación celular. Hemos observado que la adición rápida de etanol provoca la lisis celular que resulta en una disminución importante de la clasificación eficiencia. Para obtener una suspensión de células 3 ml, se deben añadir 9 ml de etanol en aproximadamente 1 min. </li><li> Se incuban las células en hielo durante 15 min con mezcla ocasional por inversión. </li><li> Suspensión de células Centrifugar a 800 xg durante 5 min y retirar la claraflotante. </li><li> Resuspender suavemente las células germinales fijos en 2 ml de tampón de clasificación. </li><li> Añadir 4 l de tinte de ADN SYTO16 (solución 1 mM en DMSO) y se incuba durante al menos 30 min (protegido de la luz). Nota: Los mejores resultados de clasificación se obtienen utilizando SYTO16 ya que es un tinte de ADN permeable que se incorpora fácilmente en los núcleos altamente compactadas de espermátidas. </li></ol> Clasificador 3. Cell Set Up Se utiliza 20-parámetro clasificador celular BDFACSAria III Aquí, un 4-láser (rojo 405 nm - violeta, 488 nm - azul, 561 nm - - amarillo-verde, 633 nm): Nota. El software BD FACSDiva 6.1.3 se utiliza para visualizar y analizar los datos. Los ajustes pueden variar en función del tipo de clasificador utilizado. <ol><li> Esterilizar el clasificador FACS mediante la ejecución de 70% de etanol como fluido envolvente durante el procedimiento de fluidos de apagado (menú &quot;citómetro&quot; en el software) antes de la clasificación. Seleccione &quot;citómetro&quot; en la barra de herramientas del software y elija &quot;sh neumáticoutdown &quot;. Siga los pasos mencionados por los procesos. </li><li> Preparar y filtrar 1x PBS con un filtro de 0,22 micras para eliminar cualquier cristal y contaminantes. Llenar el depósito de la vaina a la línea de soldadura superior en el interior del tanque. </li><li> Inicie el software e ingrese. Inicie el clasificador FACS para calentar el láser durante al menos 30 minutos antes de la clasificación. Ejecutar dos procesos de inicio de fluidos para eliminar el etanol a partir de los fluidos. Seleccione &quot;citómetro&quot; en la barra de herramientas del software y elija &quot;arranque neumático&quot;. Siga los pasos mencionados por los procesos. Nota: Esto restaura fluidos con fluido envolvente normal (1x PBS). </li><li> Limpie el bloque de clasificación y las placas de deflexión con agua destilada y secar a fondo. Sonicar una boquilla de 100 micras colocó en un tubo de agua destilada durante 2 min en un baño de sonicación. Limpie la boquilla a fondo. </li><li> Inserte la boquilla de 100 micras en la celda de flujo y gire la palanca de la boquilla de bloqueo de las agujas del reloj a la 12posición h. Ajuste la presión de funda a 20 psi. </li><li> Encienda la corriente y ajustar la amplitud de obtener valores objetivo para la frecuencia (alrededor de 30), la gota 1 (alrededor de 150) y Gap (alrededor de 12) y establecer un patrón breakoff gotita estable (gotas pocos satélite). Apague la atenuación. </li><li> Asegúrese de que la corriente es recta y golpea el centro del aspirador de residuos cambiando el ángulo del bloque tipo. Nota: Una boquilla de 130 micras, con 10 psi también se ensayó y no se encontró ninguna diferencia obvia. </li><li> Establecer retraso láser a 0 para el -77,39 azul, para el rojo, 37,33 para el violeta y -39.85 por los láseres de color amarillo-verde. </li><li> Establecer zonas de escala a 1,14 para el 1.0 azul, para el rojo, 0,75 para el violeta y 0,96 para los láseres de color amarillo-verde. </li><li> El uso de un Ordenar de prueba (en la ventana de &quot;Side Stream&quot; del software), asegúrese de que las corrientes laterales no están Fanning. En la ventana &quot;Side Stream&quot;, ajuste los controles deslizantes de tensión de cada corriente lateral para que lleguen al middle de tubo de recogida de prueba. Si la corriente de centro no es apretado, ajustar el 2º, 3º y 4 º ajustes de caída para limitar la corriente central. </li><li> Inicie el programa de instalación citómetro y Seguimiento (CS &amp; T) aplicación en el (menú &quot;citómetro&quot;) de software. </li><li> Utilice la configuración y el seguimiento de cuentas de citómetro a 1 gota por cada 350 l para automatizar la caracterización y el seguimiento de la actuación citómetro siguiendo las instrucciones del fabricante. </li><li> Salga de la aplicación CS &amp; T. Aplicar configuración de CS &amp; T, asegúrese de que los parámetros de la corriente todavía permiten un patrón breakoff gotita estable, y encienda el botón de Sweet Spot como se describe en las instrucciones del fabricante (en la &quot;ventana Breakoff&quot;). </li><li> Optimizar el valor gota Delay usando perlas fluorescentes (véase la Tabla de Materiales). <ol><li> Instale los tubos de recogida en el soporte. Abra la puerta del bloque de tipo. En la ventana &quot;Side Stream&quot;, active la tensión a continuación, seleccione &quot;Prueba de tipo &quot;y haga clic en el botón aspirador cajón para abrir el cajón y tener acceso a los tubos de recogida. </li><li> Optimizar las corrientes laterales así que van hacia el centro del tubo. Ajustar valores y deslizadores, según sea necesario. </li><li> Cierre la puerta del bloque de clase y asegúrese de ajustar el micrómetro para obtener el punto de perlas más brillantes en el centro. Cajón de residuos Deseleccionar, prueba de tipo y la tensión. Retire los tubos de recogida. </li><li> Optimizar el retraso caída de acuerdo con las instrucciones del fabricante. <ol><li> En pocas palabras, agitar vigorosamente el vial de cuentas y diluir 1 gota de los granos en 0,5 ml de PBS. </li><li> Usando las instrucciones del fabricante, establecer el retraso gota usando la plantilla experimento de la gota de retardo disponibles en el software. También puede utilizar la función Auto gota retardo para ajustar el retardo de caída. </li><li> Cargar el tubo de perlas fluorescentes y ajustar la velocidad de flujo hasta que la tasa de umbral es de ~ 3.000 a 4.000 acontecimientos / segundo. Establecer la precisión de clasificación para &quot;Inicial&quot; y últimamente a &quot;Sintonía fina&quot; en la ventana &quot;Ordenar diseño&quot;. Seleccione el filtro óptico y clasificar los granos. Ajuste el retardo gota hasta ~ 100% están ordenadas a la izquierda. Nota: Este paso es crítico para asegurarse de que las células de interés especie en una corriente lateral. Las perlas y el filtro óptico se utilizan para lograr cerca de una desviación de gotas 100%. </li></ol></li></ol></li><li> Coloque un filtro ND (densidad neutra) FSC 1.5 en el extremo izquierdo del bloque detector FSC. Establecer la extensión ventana al 2,00 microsegundo y el escalado FSC Área de 1.00 (en la pestaña de &quot;láser&quot; en la ventana &quot;citómetro&quot;). </li><li> Antes de cargar el tubo de la muestra, coloque una línea de filtro de muestra de 50 micras, con el final de la línea de muestreo para evitar la formación de grumos. Para ello, seleccione &quot;Cambiar Filtro de ejemplo&quot; en el menú &quot;citómetro&quot;. </li><li> Prueba de la composición de la muestra de fluido para lograr una buena separación entre avivar sin corriente lateral y para evitar que las células se peguen a la polypropyletubos ne. <ol><li> Cargue el tubo de la muestra, encender las placas de deflexión y seleccione &quot;prueba tipo&quot; con el cajón cerrado. Mira arroyos secundarios si se produce la separación sin demasiado Fanning. Nota: Fanning es probablemente debido a una alta concentración de proteína en el tampón de muestra. </li></ol></li><li> Cargar el tubo de muestra en el instrumento FACS. Seleccionar una muestra de agitación 300 rpm y una temperatura de muestra de 4 ° C. Seleccione &quot;Adquirir de datos&quot; en la &quot;Adquisición Dashboard&quot;. </li><li> Si es necesario, diluir la muestra para lograr una tasa de eventos apropiado para un máximo de 5.000 eventos / seg (con un caudal máximo de 3,0). Estrictamente respetar estos límites con el fin de mantener la pureza de las células clasificadas. </li><li> Para analizar y clasificar las células, optimizar el valor umbral de FSC para eliminar los escombros sin interferir en la población de interés. En escala logarítmica, ajustar el voltaje del detector de tubo fotomultiplicador (PMT) con el filtro de 530/30 a fin de hacerasegurarse de que la señal de fluorescencia de la población total se ajusta dentro de la escala, y la puerta en la población positiva (Figura 1, puerta 1). </li><li> Producir un Forward Scatter-zona (FSC-A) / dispersión lateral del área (SSC-A) parcela de la población positiva SYTO16 y ajustar el FSC-A a alrededor de 110 V en la escala lineal y la SSC-A a alrededor de 150 V en escala logarítmica para visualizar todos los eventos excluyendo muy grandes células y agregados de células. </li><li> En la ventana &quot;Ordenar Diseño&quot;, establecer el &quot;modo de ordenación de precisión&quot; a &quot;4 vías pureza&quot; (máscara Rendimiento: 0, Pureza Máscara: 32, Máscara Fase: 0). Seleccione &quot;Continuo&quot; en el menú &quot;Eventos objetivo&quot; para la clasificación continua. </li><li> Añadir poblaciones para ordenar en el campo correspondiente a los tubos (en la ventana &quot;Ordenar Layout&quot;). Coloque las poblaciones con frecuencias más altas en el centro y los que tienen frecuencias más bajas en los extremos. Establecer las placas de voltaje a 2500 V. Durante la clasificación, mantener la recolección de la muestra tUBE en el hielo. </li></ol> 4. Cell Sorting <ol><li> Inmediatamente antes de la clasificación, las células de filtro utilizando un filtro de 50 micras en un 5 ml previamente recubierto de polipropileno ronda tubo inferior. </li><li> Lave el filtro extensamente con 1-2 ml de tampón de clasificación. </li><li> Células germinales Ordenar fijos utilizando un 488 nm láser equipado clasificador de células siguiendo el esquema gating se detalla en la Figura 1. Recoger fracciones purificadas en 5 ml de polipropileno tubos de fondo redondo recubiertas previamente en la sección 1 en el hielo. <ol><li> Asegúrese de que de 100-200 l de FBS se mantiene en los tubos de recogida para evitar la adherencia de las células clasificadas a la pared del tubo. </li><li> Asegúrese de que la concentración de SYTO16 está saturando de evitar las fluctuaciones de fluorescencia durante el período de clasificación. SYTO16 está saturando cuando no hay más variación de intensidad de la tinción de células en el parámetro 488 (tinción de ADN) Alexa Fluor en los gráficos de clasificación. Si es necesario, añadir 1 l deSYTO16 al tubo de clasificación para alcanzar la saturación. </li><li> Mostrar los eventos totales en un gráfico que muestra los siguientes parámetros: número de eventos vs Alexa Fluor 488-A. </li><li> Seleccione las celdas de tinción de ADN positivos con la Puerta 1, como se muestra en la Figura 1. <ol><li> Mostrar las células de la puerta 1 en un gráfico utilizando SSC-A vs FSC-A como parámetros <ol><li> Seleccione las celdas con la puerta 2, como se muestra en la Figura 1. </li><li> Mostrar las células de la puerta 2 en un gráfico usando FSC-A vs Alexa Fluor 488-A como parámetros. </li><li> Seleccionar celdas con Puerta 5 y 6 en la Puerta Puerta 2 gráfico, como se muestra en la Figura 1. </li><li> Mostrar separado Puerta 5 y 6 en la Puerta de gráficos usando Alexa Fluor 488-W vs Alexa Fluor 488-A como parámetros. </li><li> Seleccione espermiogénesis pasos 1-9 espermátidas en la Puerta 5 gráfico y espermiogénesis pasos 10-12 espermátidas en la puerta 6 gráficos, como se muestra en la Figura 1. </li></ol></li><li> Monitorlas células de la puerta 1 en un gráfico utilizando FSC-A vs Alexa Fluor 488-A como parámetros <ol><li> Seleccionar las células con la puerta 3 y Puerta 4, como se muestra en la Figura 1. </li><li> Mostrar separado Puerta 3 y la puerta 4 de gráficos con Alexa Fluor 488-W vs Alexa Fluor 488-A como parámetros. </li><li> Seleccione espermiogénesis pasos 13-14 espermátidas en el gráfico de la Puerta 3 y espermiogénesis pasos 15-16 espermátidas en el gráfico de la puerta 4, como se muestra en la Figura 1. </li></ol></li></ol></li></ol></li></ol>

<ol>
	<li>Sato, T., 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=In+vitro+production+of+functional+sperm+in+cultured+neonatal+mouse+testes.">In vitro production of functional sperm in cultured neonatal mouse testes.</a> Nature. 471 (7339), 504-507 (2011).</li><li>Sato, T., 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=In+vitro+production+of+fertile+sperm+from+murine+spermatogonial+stem+cell+lines.">In vitro production of fertile sperm from murine spermatogonial stem cell lines.</a> Nat. Commun. 2, 472 (2011).</li><li>Sun, X., Kovacs, T., Hu, Y. -. J., Yang, W. -. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+actin+and+myosin+during+spermatogenesis.">The role of actin and myosin during spermatogenesis.</a> Mol. Biol. Rep. 38 (6), 3993-4001 (2011).</li><li>Cheng, C. Y., Mruk, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+junction+dynamics+in+the+testis:+Sertoli-germ+cell+interactions+and+male+contraceptive+development.">Cell junction dynamics in the testis: Sertoli-germ cell interactions and male contraceptive development.</a> Physiol. Rev. 82, 825-874 (2002).</li><li>Laiho, A., Kotaja, N., Gyenesei, A., Sironen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome+profiling+of+the+murine+testis+during+the+first+wave+of+spermatogenesis.">Transcriptome profiling of the murine testis during the first wave of spermatogenesis.</a> PLoS ONE. 8 (4), (2013).</li><li>Leduc, F., Maquennehan, V., Nkoma, G. B., Boissonneault, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+damage+response+during+chromatin+remodeling+in+elongating+spermatids+of+mice.">DNA damage response during chromatin remodeling in elongating spermatids of mice.</a> Biol. Reprod. 78 (2), 324-332 (2008).</li><li>Meistrich, M. L., Mohapatra, B., Shirley, C. R., Zhao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+transition+nuclear+proteins+in+spermiogenesis.">Roles of transition nuclear proteins in spermiogenesis.</a> Chromosoma. 111 (8), 483-488 (2003).</li><li>Gr&#233;goire, M. -. C., 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=Male-driven+de+novo+mutations+in+haploid+germ+cells.">Male-driven de novo mutations in haploid germ cells.</a> Mol. Hum. Reprod. 19 (8), 495-499 (2013).</li><li>Simard, 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=Instability+of+trinucleotidic+repeats+during+chromatin+remodeling+in+spermatids.">Instability of trinucleotidic repeats during chromatin remodeling in spermatids.</a> Hum. Mutat. , (2014).</li><li>Wykes, S. M., Krawetz, S. <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+spermatogenic+cells+from+adult+transgenic+mouse+testes+using+unit-gravity+sedimentation.">Separation of spermatogenic cells from adult transgenic mouse testes using unit-gravity sedimentation.</a> Mol. Biotechnol. 25 (2), 131-138 (2003).</li><li>Yoshida, S., 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=The+first+round+of+mouse+spermatogenesis+is+a+distinctive+program+that+lacks+the+self-renewing+spermatogonia+stage.">The first round of mouse spermatogenesis is a distinctive program that lacks the self-renewing spermatogonia stage.</a> Development (Cambridge, England). 133 (8), 1495-1505 (2006).</li><li>Moreno, R. D., Lizama, C., Urz&#250;a, N., Vergara, S. P., Reyes, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspase+activation+throughout+the+first+wave+of+spermatogenesis+in+the+rat.">Caspase activation throughout the first wave of spermatogenesis in the rat.</a> Cell Tissue Res. 325 (3), 533-540 (2006).</li><li>Meistrich, M. L., Trostle-Weige, P. K., Van Beek, M. E. <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+specific+stages+of+spermatids+from+vitamin+A-synchronized+rat+testes+for+assessment+of+nucleoprotein+changes+during+spermiogenesis.">Separation of specific stages of spermatids from vitamin A-synchronized rat testes for assessment of nucleoprotein changes during spermiogenesis.</a> Biol. Reprod. 51 (2), 334-344 (1994).</li><li>van Pelt, A. M., de Rooij, D. G. <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+the+seminiferous+epithelium+after+vitamin+A+replacement+in+vitamin+A-deficient+mice.">Synchronization of the seminiferous epithelium after vitamin A replacement in vitamin A-deficient mice.</a> Biol. Reprod. 43, 363-367 (1990).</li><li>Getun, I. V., Torres, B., Bois, P. R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+purification+of+mouse+meiotic+cells.">Flow cytometry purification of mouse meiotic cells.</a> J. Vis. Exp. , (2011).</li><li>Kovtun, I. V., McMurray, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trinucleotide+expansion+in+haploid+germ+cells+by+gap+repair.">Trinucleotide expansion in haploid germ cells by gap repair.</a> Nature Genet. 27 (4), 407-411 (2001).</li><li>Bastos, H., 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=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 (1), 40-49 (2005).</li><li>Lassalle, B., Ziyyat, A., Testart, J., Finaz, C., Lef&#232;vre, A. <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+method+to+isolate+round+spermatids+from+mouse+testis.">Flow cytometric method to isolate round spermatids from mouse testis.</a> Hum. Reprod. 14 (2), 388-394 (1999).</li></ol>

The authors declare no competing financial interests.

Células de espermatogénesis siempre han sido difíciles de estudiar debido a la complejidad del epitelio seminífero, así como el éxito limitado de cultivo in vitro. A través de los años, muchos enfoques para purificar se desarrollaron las células germinales a partir de diversas especies. Utilizando técnicas de sedimentación de purificación de la gravedad con Percoll o gradientes de albúmina de suero bovino por lo general proporcionan un buen rendimiento de células germinales intactas, pero carecen ...

Haploid round spermatids differentiate into spermatozoa by a process called spermiogenesis. This involves many different steps including the acquisition of a flagellum, chromatin and cytoskeleton remodeling, condensation of the nucleus as well as the loss of most of the cytoplasm. These unique cellular events must be finely regulated in order to produce a mature functional gamete with an intact genome suitable for fertilization. Spermiogenesis can hardly be studied in vitro since no reliable cell culture system has so far been able to support progression through the different steps of the process. Moreover, actual in vitro techniques lead to a poor yield1,2. In vivo, proper transitions through the different steps of spermiogenesis are crucial for the natural functional integrity of the male gamete. Successful purification of spermatids according to their differentiation steps has never been accomplished with a level of enrichment sufficient to allow molecular characterization of spermiogenesis. For instance, purification of key steps of the spermatidal differentiation would be especially useful to study the developing acrosome, formation of the midpiece3, cell junction dynamics4, RNA dynamics5, chromatin remodeling process6,7 or genomic stability8. Purification of spermatids has been hampered by their progressive morphological transformation, the lack of known stage-specific external biomarkers, and their peculiar shape and size. 
Although most male germ cells display a direct relationship between DNA staining and ploidy (DNA content), we noticed that such positive correlation is no longer applicable to spermatids. This stems from our early observation that seminiferous tubule sections show variable intensity of DNA staining throughout the different spermiogenesis steps. Although DNA staining is consistent with their haploid set of chromosomes from spermiogenesis steps 1 to 7 (round spermatids), we observed a sharp increase in fluorescence intensity with DAPI or SYTO 16 around the onset of nuclear reorganization and chromatin remodeling (spermiogenesis step 8) reaching a peak at the onset of nuclear condensation (spermiogenesis steps 11-12). Following condensation of the nucleus, DNA staining intensity decreases until spermiation (spermiogenesis step 16). We surmised that this was likely associated with the formation of their peculiar chromatin structure transition where histones are replaced by protamines. We therefore developed a reliable flow cytometry method that allows the separation of spermatids using the variation of DNA intensity of spermatids as a main selection parameter.
A simple flow cytometry approach is described to separate mouse spermatids with high purity (95-100%) based on their apparent DNA content (SYTO16 staining), size and granulosity. Spermatids are separated into four populations; spermiogenesis steps 1-9, 10-12, 13-14 and 15-16. Purified spermatids are suitable for genetic/genomic analysis, as well as proteomic applications as described in a recent publication from our group9.

<table border="0" cellpadding="0" cellspacing="0" width="565">
	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">Isoflurane</td>
			<td style="width:119px;">ABBOT</td>
			<td style="width:129px;">05260-05</td>
			<td style="width:184px;">For mouse anesthesia before euthanasia</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">Fetal bovine serum</td>
			<td style="width:119px;">Wisent</td>
			<td style="width:129px;">90150</td>
			<td style="width:184px;">For tube coating</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:135px;">1X PBS</td>
			<td style="width:119px;"></td>
			<td style="width:129px;"></td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">EDTA</td>
			<td style="width:119px;">BioShop</td>
			<td style="width:129px;">EDT</td>
			<td style="width:184px;">For sorting buffer preparation</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">HEPES</td>
			<td style="width:119px;">Sigma</td>
			<td style="width:129px;">H</td>
			<td style="width:184px;">For sorting buffer preparation</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">100 % Ethanol</td>
			<td style="width:119px;">Les alcools de commerce</td>
			<td style="width:129px;">092-09-11N</td>
			<td style="width:184px;">For cell fixation</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">SYTO 16</td>
			<td style="width:119px;">Life Technologies</td>
			<td style="width:129px;">S7578</td>
			<td style="width:184px;">DNA staining</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:135px;">5 ml polypropylene round bottom tubes</td>
			<td style="width:119px;">BD Falcon</td>
			<td style="width:129px;">352063</td>
			<td style="width:184px;">Sorted cells collection</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:135px;">15 ml polypropylene conical bottom tubes</td>
			<td style="width:119px;">PROgene</td>
			<td style="width:129px;">1500</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:135px;">50 ml polypropylene conical bottom tubes</td>
			<td style="width:119px;">PROgene</td>
			<td style="width:129px;">5000</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:135px;">TEC4 anaesthetic vaporizer</td>
			<td style="width:119px;">Ohmeda</td>
			<td style="width:129px;">1160526</td>
			<td style="width:184px;">For mouse euthanasia</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:135px;">CO2 gas tank</td>
			<td style="width:119px;">Praxair</td>
			<td style="width:129px;">C799117902</td>
			<td style="width:184px;">For mouse euthanasia</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:135px;">O2 gas tank</td>
			<td style="width:119px;">Praxair</td>
			<td style="width:129px;">O254130501</td>
			<td style="width:184px;">For mouse euthanasia</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:135px;">Homemade mouse gas chamber</td>
			<td style="width:119px;"></td>
			<td style="width:129px;"></td>
			<td style="width:184px;">For mouse euthanasia</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">40 &#181;m Falcon cell strainer</td>
			<td style="width:119px;">Corning Incorporated</td>
			<td style="width:129px;">352340</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">50-micron sample line filters</td>
			<td style="width:119px;">BD Biosciences</td>
			<td style="width:129px;">649049</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:135px;">Vortex mixer</td>
			<td style="width:119px;">Labnet international, inc.</td>
			<td style="width:129px;">S0200</td>
			<td style="width:184px;">For cell fixation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:135px;">Dynac centrifuge</td>
			<td style="width:119px;">Clay Adams</td>
			<td style="width:129px;">101</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">Celltrics 50 &#181;m filters</td>
			<td style="width:119px;">Partec</td>
			<td style="width:129px;">04-004-2327</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:135px;">488 nm laser-euipped cell sorter</td>
			<td style="width:119px;">BD Biosciences</td>
			<td style="width:129px;">FACSAria III</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:135px;">Accudop Fluorescent Beads</td>
			<td style="width:119px;">BD Biosciences</td>
			<td style="width:129px;">345249</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:135px;">Sorting Buffer: 1X PBS, 1mM EDTA pH 8.0, 25mM HEPES pH 7.0, 1%FBS</td>
			<td></td>
			<td></td>
			<td style="width:184px;">FBS is heat-inactivated. Make fresh solution, 0.22 &#956;m filtered and keep at 4&#176;C.</td>
		</tr>
	</tbody>
</table>

step-specific sorting of mouse spermatids by flow cytometry

La diferenciación de espermátidas de ratón es uno proceso crítico para la producción de un gameto masculino funcional con un genoma intacto para ser transmitido a la siguiente generación. Hasta ahora, los estudios moleculares de esta transición morfológica se han visto obstaculizados por la falta de un método que permite una separación adecuada de estos pasos importantes de la diferenciación espermátide para análisis posteriores. Los anteriores intentos de la selección adecuada de estas células usando citometría de flujo pueden haber sido difícil debido a un aumento en la fluorescencia peculiar ADN en espermátidas sometidos a remodelación de la cromatina. Sobre la base de esta observación, proporcionamos los detalles de un flujo sencillo citometría de esquema, lo que permite la purificación reproducible de cuatro poblaciones de espermátidas de ratón fijados con etanol, cada uno representando un estado diferente en el proceso de remodelación nuclear. Enriquecimiento de Población se confirma el uso de marcadores de pasos específicos y criterios morfológicos. Las espermátidas purificadas se pueden utilizar para genómico y proteomic analiza.

Estrategia de apertura de puerta se usa con citometría de flujo La Figura 1 representa la estrategia gating utilizado en citometría de flujo para clasificar cuatro poblaciones spermatid altamente puros. Brevemente, las células con tinción de ADN positivo (Alexa Fluor 488-A) se seleccionan en primer lugar con la Puerta 1. Espermátidas de espermiogénesis pasos son seleccionados 1-12 (puerta 2) en un gráfi...

Watch this Scientific Journal Video about Step-specific Sorting of Mouse Spermatids by Flow Cytometry at JoVE.com

Step-specific Sorting of Mouse Spermatids by Flow Cytometry

We describe a sorting strategy for mouse spermatids using flow cytometry. Spermatids are sorted into four highly pure populations, including round (spermiogenesis steps 1-9), early elongating (spermiogenesis steps 10-12), late elongating (spermiogenesis steps 13-14) and elongated spermatids (spermiogenesis steps 15-16). DNA staining, size and granulosity are used as selection parameters.

-Paso específica Clasificación de Ratón Espermátidas por Citometría de Flujo

The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to ...

step-specific-sorting-of-mouse-spermatids-by-flow-cytometry

Research

JoVE Journal

Developmental Biology

10.4K vistas.  Université de Sherbrooke. We describe a sorting strategy for mouse spermatids using flow cytometry. Spermatids are sorted into four highly pure populations, including round (spermiogenesis steps 1-9), early elongating (spermiogenesis steps 10-12), late elongating (spermiogenesis steps 13-14) and elongated spermatids (spermiogenesis steps 15-16). DNA staining, size and granulosity are used as selection parameters.

Video: Step-specific Sorting of Mouse Spermatids by Flow Cytometry

Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where they act as front line sentinels of infection and tissue damage. While other immune cells are continuously renewed from hematopoietic stem and progenitor cells (HSPC) located in the bone marrow, a lineage of macrophages, known as resident macrophages, have been shown to be self-maintained in tissues without input from bone marrow HSPCs. This lineage is exemplified by microglia in the brain, Kupffer cells in the liver and Langerhans cells in the epidermis among others. The intestinal and colon lamina propria are the only adult tissues devoid of HSPC-independent resident macrophages. Recent investigations have identified that resident macrophages originate from the extra-embryonic yolk sac hematopoiesis from progenitor(s) distinct from fetal hematopoietic stem cells (HSC). Among yolk sac definitive hematopoiesis, erythromyeloid progenitors (EMP) give rise both to erythroid and myeloid cells, in particular resident macrophages. EMP are only generated within the yolk sac between E8.5 and E10.5 days of development and they migrate to the fetal liver as early as circulation is connected, where they expand and differentiate until at least E16.5. Their progeny includes erythrocytes, macrophages, neutrophils and mast cells but only EMP-derived macrophages persist until adulthood in tissues. The transient nature of EMP emergence and the temporal overlap with HSC generation renders the analysis of these progenitors difficult. We have established a tamoxifen-inducible fate mapping protocol based on expression of the macrophage cytokine receptor Csf1r promoter to characterize EMP and EMP-derived cells in vivo by flow cytometry.

While infiltrating macrophages are continuously recruited to adult tissues from circulating precursors, resident macrophages seed their tissue during development, where they are maintained without further input from progenitors. The progenitors for resident macrophages were recently identified. Here, we present methods for the genetic fate mapping of the resident macrophage progenitors.

Identificación de progenitores eritromioideos y su progenie en el embrión de ratón por citometría de flujo

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where ...

Investigación

Biología del Desarrollo

Isolating and Analyzing Cells of the Pancreas Mesenchyme by Flow Cytometry

The pancreas is comprised of epithelial cells that are required for food digestion and blood glucose regulation. Cells of the pancreas microenvironment, including endothelial, neuronal, and mesenchymal cells were shown to regulate cell differentiation and proliferation in the embryonic pancreas. In the adult, the function and mass of insulin-producing cells were shown to depend on cells in their microenvironment, including pericyte, immune, endothelial, and neuronal cells. Lastly, changes in the pancreas microenvironment were shown to regulate pancreas tumorigenesis. However, the cues underlying these processes are not fully defined. Therefore, characterizing the different cell types that comprise the pancreas microenvironment and profiling their gene expression are crucial to delineate the tissue development and function under normal and diseased states. Here, we describe a method that allows for the isolation of mesenchymal cells from the pancreas of embryonic, neonatal, and adult mice. This method utilizes the enzymatic digestion of mouse pancreatic tissue and the subsequent fluorescence-activated cell sorting (FACS) or flow-cytometric analysis of labeled cells. Cells can be labeled by either immunostaining for surface markers or by the expression of fluorescent proteins. Cell isolation can facilitate the characterization of genes and proteins expressed in cells of the pancreas mesenchyme. This protocol was successful in isolating and culturing highly enriched mesenchymal cell populations from the embryonic, neonatal, and adult mouse pancreas.

Here, we describe a method for the isolation of cells in the pancreas microenvironment from embryonic, neonatal and adult mouse tissue, focusing on the isolation of mesenchymal cells. This method allows profiling of cell gene expression and protein secretion in order to elucidate the extrinsic signals that regulate pancreas development, function, and tumorigenesis.

Aislar y analizar células del páncreas mesénquima por Citometría de Flujo

The pancreas is comprised of epithelial cells that are required for food digestion and blood glucose regulation. Cells of the pancreas microenvironment, ...

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. Currently, it allows the discrimination of up to 9 murine germ cell populations with high yield and purity. This high-resolution in discrimination and purification is possible due to unique changes in chromatin structure and quantity throughout spermatogenesis. These patterns can be captured by flow cytometry of male germ cells stained with fluorescent DNA-binding dyes such as Hoechst-33342 (Hoechst). Herein is a detailed description of a recently developed protocol to isolate mammalian testicular germ cells. Briefly, single cell suspensions are generated from testicular tissue by mechanical dissociation, double stained with Hoechst and propidium iodide (PI) and processed by flow cytometry. A serial gating strategy, including the selection of live cells (PI negative) with different DNA content (Hoechst intensity), is used during FACS sorting to discriminate up to 5 germ cell types. These include, with corresponding average purities (determined by microscopy evaluation): spermatogonia (66%), primary (71%) and secondary (85%) spermatocytes, and spermatids (90%), further separated into round (93%) and elongating (87%) subpopulations. Execution of the entire workflow is straightforward, allows the isolation of 4 cell types simultaneously with the appropriate FACS machine, and can be performed in less than 2 h. As reduced processing time is crucial to preserve the physiology of ex vivo cells, this method is ideal for downstream high-throughput studies of male germ cell biology. Moreover, a standardized protocol for multispecies purification of mammalian germ cells eliminates methodological sources of variables and allows a single set of reagents to be used for different animal models.

This work describes the standardization of a method to obtain purified germ cell populations from testicular tissue of different mammalian species. It is a straightforward protocol that combines mechanical testis dissociation, staining with Hoechst-33342 and propidium iodide, and FACS sorting, with wide applications in comparative studies of male reproductive biology.

Un enfoque estandarizado para la purificación multiespecífica de células germinales masculinas de mamíferos por disociación mecánica de tejidos y citometría de flujo

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. ...

Use of Hematopoietic Stem Cell Transplantation to Assess the Origin of Myelodysplastic Syndrome

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood cytopenias, dysplasia, and a propensity for transformation to acute leukemia. NUP98-HOXD13 (NHD13) transgenic mice recapitulate human MDS in terms of peripheral blood cytopenias, dysplasia, and transformation to acute leukemia. We previously demonstrated that MDS could be transferred from a genetically engineered mouse with MDS to wild-type recipients by transplanting MDS bone marrow nucleated cells (BMNC). To more clearly understand the MDS cell of origin, we have developed approaches to transplant specific, immunophenotypically defined hematopoietic subsets. In this article, we describe the process of isolating and transplanting specific populations of hematopoietic stem and progenitor cells. Following transplantation, we describe approaches to assess the efficiency of transplantation and persistence of the donor MDS cells.

We describe the use of hematopoietic stem cell transplantation (HSCT) to assess the malignant potential of genetically engineered hematopoietic cells. HSCT is useful for evaluating various malignant hematopoietic cells in vivo as well as generating a large cohort of mice with myelodysplastic syndromes (MDS) or leukemia to evaluate novel therapies.

Uso de trasplante de células madre hematopoyéticas para evaluar el origen del síndrome mielodisplásico

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood ...

Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a method whereby live-cell flow cytometry is extended upon to analyze the multiple functions of P2X7 receptor activation in real-time. Using a time module installed on a flow cytometer, live-cell functionality can be assessed and plotted over a given time period to explore the kinetics of calcium influx, transmembrane pore formation, and phagocytosis. This simple method is advantageous as all three canonical functions of the P2X7 receptor can be assessed using one machine, and the gathered data plotted over time provides information on the entire live-cell population rather than single-cell recordings often obtained using technically challenging patch-clamp methods. Calcium influx experiments use a calcium indicator dye, while P2X7 pore formation assays rely on ethidium bromide being allowed to pass through the transmembrane pore formed upon high agonist concentrations. Yellow-green (YG) latex beads are utilized to measure phagocytosis. Specific agonists and antagonists are applied to investigate the effects of P2X7 receptor activity. Individually, these methods can be modified to provide quantitative data on any number of calcium channels and purinergic and scavenger receptors. Taken together, they highlight how the use of real-time live-cell flow cytometry is a rapid, cost-effective, reproducible, and quantifiable method to investigate P2X7 receptor function.

Providing single-cell sensitivity, real-time flow cytometry is uniquely suited to quantify multimodal receptor functions of live cultures. Using adult neural progenitor cells, the P2X7 receptor function was assessed via calcium influx detected by calcium indicator dye, transmembrane pore formation by ethidium bromide uptake, and phagocytosis using fluorescent latex beads.

Citometría de flujo en tiempo real de células vivas para investigar la afluencia del calcio, formación de poro y fagocitosis por los receptores P2X7 en células progenitoras neurales adultas

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a ...

Isolation and Staining of Mouse Skin Keratinocytes for Cell Cycle Specific Analysis of Cellular Protein Expression by Mass Cytometry

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the epidermis of mouse skin. There are seven important steps: separation of the epidermis from the dermis, digestion of the epidermis, staining of the epidermal cell populations with cisplatin, sample barcoding, staining with metal tagged antibodies for cell cycle markers and proteins of interest, detection of metal-tagged antibodies by mass cytometry, and the analysis of expression in the various cell cycle phases. The advantage of this approach over histological methods is the potential to assay the expression pattern of &#62;40 different markers in a single cell at different phases of the cell cycle. This approach also allows for the multivariate correlation analysis of protein expression that is more quantifiable than histological/imaging methods. The disadvantage of this protocol is that a suspension of single cells is needed, which results in the loss of location information provided by the staining of tissue sections. This approach may also require the inclusion of additional markers to identify different cell types in crude cell suspensions. The application of this protocol is evident in the analysis of hyperplastic skin disease models. Moreover, this protocol can be adapted for the analysis of specific sub-type of cells (e.g., stem cells) by the addition of lineage-specific antibodies. This protocol can also be adapted for the analysis of skin cells in other experimental species.

This protocol describes how to isolate skin keratinocytes from mouse models, to stain with metal-tagged antibodies, and to analyze stained cells by mass cytometry in order to profile the expression pattern of proteins of interest in the different cell cycle phases.

Aislamiento y tinción de queratinocitos de piel de ratón para el análisis específico del ciclo celular de la expresión de proteínacelular espora

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the ...

Enrichment of Pachytene Spermatocytes and Spermatids from Mouse Testes Using Standard Laboratory Equipment

To characterize each step of spermatogenesis, researchers must separate different subpopulations of germ cells from testes. However, isolating discrete populations is challenging, because the adult testis contains a complex mix of germ cells from all steps of spermatogenesis along with certain populations of somatic cells. Over the past few decades, different techniques such as centrifugal elutriation, fluorescence-activated cell sorting (FACS), and STA-PUT have been successfully applied to the isolation of germ cells. A drawback is that they all require dedicated devices and specialized training. Following principles underlying the STA-PUT method, a simple protocol has been developed for the isolation of pachytene spermatocytes, round spermatids, and elongating spermatids from mouse testes. After preparing a single cell suspension of testicular cells, specific cell populations are enriched by gravity sedimentation through a discontinuous bovine serum albumin (BSA) density gradient. The cell fractions are then manually collected and microscopically analysed. This modified density gradient for round spermatids (MDR) sedimentation protocol can be widely applied, because it requires only standard laboratory equipment. Furthermore, the protocol requires minimal starting materials, reducing its cost and use of laboratory animals.

Presented here is a protocol for enriching pachytene spermatocytes, round spermatids, and elongating spermatids from adult mouse testes using a discontinuous bovine serum albumin density gradient with standard laboratory equipment.

Enriquecimiento de espermaocitos y espermatídos de los testículos de ratón que utilizan equipos de laboratorio estándar

To characterize each step of spermatogenesis, researchers must separate different subpopulations of germ cells from testes. However, isolating discrete ...

Using Flow Cytometry to Detect and Quantitate Altered Blood Formation in the Developing Zebrafish

The diversity of cell lineages that comprise mature blood in vertebrate animals arise from the differentiation of hematopoietic stem and progenitor cells (HSPCs). This is a critical process that occurs throughout the lifespan of organisms, and disruption of the molecular pathways involved during embryogenesis can have catastrophic long-term consequences. For a multitude of reasons, zebrafish (Danio rerio) has become a model organism to study hematopoiesis. Zebrafish embryos develop externally, and by 7 days postfertilization (dpf) have produced most of the subtypes of definitive blood cells that will persist for their lifetime. Assays to assess the number of hematopoietic cells have been developed, mainly utilizing specific histological stains, in situ hybridization techniques, and microscopy of transgenic animals that utilize blood cell-specific promoters driving the expression of fluorescent proteins. However, most staining assays and in situ hybridization techniques do not accurately quantitate the number of blood cells present; only large differences in cell numbers are easily visualized. Utilizing transgenic animals and analyzing individuals with fluorescent or confocal microscopy can be performed, but the quantitation of these assays relies on either counting manually or utilizing expensive imaging software, both of which can make errors. Development of additional methods to assess blood cell numbers would be economical, faster, and could even be automated to quickly assess the effect of CRISPR-mediated genetic modification, morpholino-mediated transcript reduction, and the effect of drug compounds that affect hematopoiesis on a large scale. This novel assay to quantitate blood cells is performed by dissociating whole zebrafish embryos and analyzing the amount of fluorescently labelled blood cells present. These assays should allow elucidation of molecular pathways responsible for blood cell generation, expansion, and regulation during embryogenesis, which will allow researchers to further discover novel factors altered during blood diseases, as well as pathways essential during the evolution of vertebrate hematopoiesis.

This assay is a simple method to quantitate hematopoietic cells in developing embryonic zebrafish. Blood cells from dissociated zebrafish are subjected to flow cytometry analysis. This allows the detection of blood defects in mutant animals and after genetic modification.

Uso de la citometría de flujo para detectar y cuantificar la formación de sangre alterada en el pez cebra en desarrollo

The diversity of cell lineages that comprise mature blood in vertebrate animals arise from the differentiation of hematopoietic stem and progenitor cells ...

FACS-Isolation and Culture of Fibro-Adipogenic Progenitors and Muscle Stem Cells from Unperturbed and Injured Mouse Skeletal Muscle

Fibro-adipogenic progenitor cells (FAPs) are a population of skeletal muscle-resident mesenchymal stromal cells (MSCs) capable of differentiating along fibrogenic, adipogenic, osteogenic, or chondrogenic lineage. Together with muscle stem cells (MuSCs), FAPs play a critical role in muscle homeostasis, repair, and regeneration, while actively maintaining and remodeling the extracellular matrix (ECM). In pathological conditions, such as chronic damage and muscular dystrophies, FAPs undergo aberrant activation and differentiate into collagen-producing fibroblasts and adipocytes, leading to fibrosis and intramuscular fatty infiltration. Thus, FAPs play a dual role in muscle regeneration, either by sustaining MuSC turnover and promoting tissue repair or contributing to fibrotic scar formation and ectopic fat infiltrates, which compromise the integrity and function of the skeletal muscle tissue. A proper purification of FAPs and MuSCs is a prerequisite for understanding the biological role of these cells in physiological as well as in pathological conditions. Here, we describe a standardized method for the simultaneous isolation of FAPs and MuSCs from limb muscles of adult mice using fluorescence-activated cell sorting (FACS). The protocol describes in detail the mechanical and enzymatic dissociation of mononucleated cells from whole limb muscles and injured tibialis anterior (TA) muscles. FAPs and MuSCs are subsequently isolated using a semi-automated cell sorter to obtain pure cell populations. We additionally describe an optimized method for culturing quiescent and activated FAPs and MuSCs, either alone or in coculture conditions.

The precise identification of fibro-adipogenic progenitor cells (FAPs) and muscle stem cells (MuSCs) is critical to studying their biological function in physiological and pathological conditions. This protocol provides guidelines for the isolation, purification, and culture of FAPs and MuSCs from adult mouse muscles.

FACS-aislamiento y cultivo de progenitores fibroadipogénicos y células madre musculares a partir de músculo esquelético de ratón imperturbable y lesionado

Fibro-adipogenic progenitor cells (FAPs) are a population of skeletal muscle-resident mesenchymal stromal cells (MSCs) capable of differentiating along ...

Identification and Isolation of Burst-Forming Unit and Colony-Forming Unit Erythroid Progenitors from Mouse Tissue by Flow Cytometry

Early erythroid progenitors were originally defined by their colony-forming potential in vitro and classified into burst-forming and colony-forming &#34;units&#34; known as BFU-e and CFU-e. Until recently, methods for the direct prospective and complete isolation of pure BFU-e and CFU-e progenitors from freshly isolated adult mouse bone marrow were not available. To address this gap, a single-cell RNA-seq (scRNAseq) dataset of mouse bone marrow was analyzed for the expression of genes coding for cell surface markers. This analysis was combined with cell fate assays, allowing the development of a novel flow cytometric approach that identifies and allows the isolation of complete and pure subsets of BFU-e and CFU-e progenitors in mouse bone marrow or spleen. This approach also identifies other progenitor subsets, including subsets enriched for basophil/mast cell and megakaryocytic potentials. The method consists of labeling fresh bone marrow or spleen cells with antibodies directed at Kit and CD55. Progenitors that express both these markers are then subdivided into five principal populations. Population 1 (P1 or CFU-e, Kit+ CD55+ CD49fmed/low CD105med/high CD71med/high) contains all of the CFU-e progenitors and may be further subdivided into P1-low (CD71med CD150high) and P1-hi (CD71high CD150low), corresponding to early and late CFU-e, respectively; Population 2 (P2 or BFU-e, Kit+ CD55+ CD49fmed/low CD105med/high CD71low CD150high) contains all of the BFU-e progenitors; Population P3 (P3, Kit+ CD55+ CD49fmed/high CD105med/low CD150low CD41low) is enriched for basophil/mast cell progenitors; Population 4 (P4, Kit+ CD55+ CD49fmed/high CD105med/low CD150high CD41+) is enriched for megakaryocytic progenitors; and Population 5 (P5, Kit+ CD55+ CD49fmed/high CD105med/low CD150high CD41-) contains progenitors with erythroid, basophil/mast cell, and megakaryocytic potential (EBMP) and erythroid/ megakaryocytic/ basophil-biased multipotential progenitors (MPPs). This novel approach allows greater precision when analyzing erythroid and other hematopoietic progenitors and also allows for reference to transcriptome information for each flow cytometrically defined population.

Here, we describe a novel flow cytometric method for prospective isolation of early burst-forming unit erythroid (BFU-e) and colony-forming unit erythroid (CFU-e) progenitors directly from fresh mouse bone marrow and spleen. This protocol, developed based on single-cell transcriptomic data, is the first to isolate all the tissue's erythroid progenitors with high purity.

Identificación y aislamiento de progenitores eritroides de unidades formadoras de ráfagas y unidades formadoras de colonias a partir de tejido de ratón mediante citometría de flujo

Early erythroid progenitors were originally defined by their colony-forming potential in vitro and classified into burst-forming and colony-forming ...