Les auteurs tiennent à remercier le Dr Leonid Volkov et Éric Bouchard pour leurs conseils techniques concernant microscopie à épifluorescence. Aide financière Financé par les Instituts de recherche en santé (subvention # MOP-93781) GB canadiennes

Les soins des animaux était en conformité avec la protection des animaux et l&#39;utilisation comité Université de Sherbrooke. 1. Préparation du tube <ol><li> Le jour avant le tri de cellules, ajouter 2.1 ml de sérum bovin foetal inactivé par la chaleur (FBS) à 5 ml polypropylene tubes à fond rond et à 15 ml et 50 ml polypropylene tubes coniques. Étape critique: Veiller à ce que chaque tube utilisé dans le protocole est revêtu. Remarque: Un revêtement FBS empêche les cellules germinales de coller aux parois du tube. </li><li> Mélangez lentement O / N par inversion à 4 ° C pour enduire en utilisant les tubes uniformément un rotateur. </li><li> Le lendemain, retirez FBS de tubes revêtus par décantation. <ol><li> * Étape critique: Veiller à ce que les 5 ml polypropylène tubes à fond rond et les 15 et 50 ml tubes utilisés pour la préparation cellulaire (protocole étapes 02.01 à 02.11) sont complètement vidés de FBS pour empêcher Fanning (ie, la déviation imprécise des courants latéraux). Utilisez une pipette P200 pour enlever les FBS. <br/&gt; Remarque: Des résidus de FBS dans des tubes avant de tri peut conduire à une perte de cellules ou de contamination d&#39;autres tubes utilisés pendant le tri de cellules, </li><li> Laisser un petit volume résiduel de FBS (environ 100-200 pi) dans la 5 ml polypropylène ronde des tubes inférieurs utilisés pour collecter les cellules purifiées. </li></ol></li></ol> 2. Préparation des cellules <ol><li> Sacrifier souris mâle sous anesthésie utilisant O 2 / isoflurane (induction à 5% puis maintenu à 2%), suivie par asphyxie au CO2. </li><li> Accise et décapsulent deux testicules et émincer avec de petits ciseaux dans 500 pi de tampon de tri dans un tube de 1,5 ml. </li><li> Rincer les cellules germinales à partir de tubes séminifères par douce pipetage de haut en bas dans le tube de 1,5 ml, d&#39;abord avec une pointe de 100-1.000 pi tronquée, puis, avec une pointe 100-1000 ul intacte. </li><li> Enlever les débris et bouquets par la première étape de filtration utilisant un tamis cellulaire de 40 um et de recueillir filtrat dans les 50 ml coniques prev tubeiously enduit avec du FBS à l&#39;étape 1. </li><li> Laver le filtre une fois avec le tri tampon jusqu&#39;à un volume total de 3 ml et le transfert filtrat dans le tube de 15 ml conique préalablement revêtu à l&#39;étape 1. </li><li> Ajouter 16 ul d&#39;EDTA 50 mM pH 8 par millilitre de suspension cellulaire (48 pi pour 3 ml) de façon à obtenir une concentration finale de 0,8 mM d&#39;EDTA. </li><li> Pour fixer les cellules germinales, ajouter lentement 3 volumes de glace-froid 100% d&#39;éthanol en utilisant une pipette de 10 ml avec une légère agitation (vortex à basse vitesse), qui va produire une suspension laiteuse. Étape cruciale: en ajoutant lentement de l&#39;éthanol est crucial et important de préserver l&#39;intégrité de la préparation cellulaire. Nous avons constaté que l&#39;addition rapide de l&#39;éthanol provoque la lyse des cellules a entraîné une diminution importante de l&#39;efficacité du tri. Pour une suspension de cellules 3 ml, 9 ml d&#39;éthanol, il convient d&#39;ajouter à environ 1 min. </li><li> Incuber les cellules sur de la glace pendant 15 minutes avec mélange occasionnel par inversion. </li><li> Centrifugeuse suspension de cellules à 800 g pendant 5 min et retirez le clairsurnageant. </li><li> Resuspendre doucement les cellules germinales fixes dans 2 ml de tampon de tri. </li><li> Ajouter 4 pi de l&#39;ADN SYTO16 colorant (solution à 1 mM dans le DMSO) et incuber pendant au moins 30 min (abri de la lumière). Remarque: Les meilleurs résultats sont obtenus en utilisant de tri SYTO16 car il est un colorant d&#39;ADN perméable qui peut être facilement incorporée dans les noyaux fortement compactées de spermatides. </li></ol> 3. trieur de cellules Set Up Remarque: Ici, un 4-laser (405 nm - violet, 488 nm - bleu, 561 nm - jaune-vert, 633 nm - rouge) 20-paramètre BDFACSAria III trieur de cellules est utilisée. Le logiciel BD FACSDiva 6.1.3 est utilisé pour visualiser et analyser les données. Les paramètres peuvent varier en fonction du type de trieur utilisé. <ol><li> Stériliser le trieur FACS en exécutant 70% d&#39;éthanol comme fluide de gaine pendant la procédure fluidique d&#39;arrêt (menu &quot;cytomètre&quot; dans le logiciel) avant le tri. Sélectionnez «cytomètre&quot; dans la barre d&#39;outils du logiciel et choisissez &quot;sh fluidiqueutdown &quot;. Suivez les étapes mentionnées par les processus. </li><li> Préparer et un filtrage 1x PBS avec un filtre de 0,22 um afin d&#39;éliminer tous les cristaux et les contaminants. Remplir le réservoir de la gaine à la ligne de soudure supérieure à l&#39;intérieur de la cuve. </li><li> Lancez le logiciel et connectez-vous. Commencez le trieur FACS pour réchauffer les lasers pendant au moins 30 min avant le tri. Exécutez deux processus de démarrage fluidiques pour débusquer l&#39;éthanol à partir de la fluidique. Sélectionnez «cytomètre&quot; dans la barre d&#39;outils du logiciel et choisissez &quot;démarrage fluidique». Suivez les étapes mentionnées par les processus. Note: Ceci restaure fluidique avec le fluide de gaine normale (1x PBS). </li><li> Nettoyez le bloc de tri et les plaques de déviation avec de l&#39;eau distillée et séchez-les soigneusement. Soniquer une buse de 100 pm placée dans un tube de l&#39;eau distillée pendant 2 min dans un bain de sonication. Essuyez soigneusement la buse. </li><li> Insérez la buse de 100 um dans la cellule d&#39;écoulement et tournez le levier de verrouillage dans le sens horaire buse à la 12position h. Régler la pression de la gaine à 20 psi. </li><li> Tournez sur le flux et ajuster l&#39;amplitude d&#39;obtenir des valeurs cibles pour la fréquence (environ 30), baisse de 1 (environ 150) et Gap (environ 12) et d&#39;établir un modèle de gouttelette breakoff stable (quelques gouttes satellites). Éteignez atténuation. </li><li> Assurez-vous que le flux est droite et frappe le centre de l&#39;aspirateur de déchets en changeant l&#39;angle du bloc de tri. Remarque: Une buse de 130 um à 10 psi a également été testé et aucune différence évidente n&#39;a été trouvée. </li><li> Définit le délai laser à 0 pour la -77,39 bleu, pour le rouge, 37.33 pour la violette et -39,85 pour les lasers jaune-vert. </li><li> Définir les zones d&#39;échelle à 1,14 pour le 1.0 bleu, pour le rouge, de 0,75 pour la violette et de 0,96 pour les lasers jaune-vert. </li><li> Utilisation d&#39;un Tri de test (dans la fenêtre &quot;Side Stream&quot; du logiciel), assurez-vous que les flux secondaires ne sont pas Fanning. Dans la fenêtre &quot;Side Stream&quot;, régler les curseurs de tension de chaque courant latéral de façon à frapper le middle de tube de collecte de test. Si le flux de centre est pas étanche, ajuster la 2 ème, 3 ème et 4 ème paramètres de chute pour limiter le flux de centre. </li><li> Démarrez le Setup cytomètre et le suivi (CS &amp; T) application dans le logiciel (menu &quot;cytomètre&quot;). </li><li> Utilisez la configuration et le suivi des perles cytomètre à 1 goutte par 350 pi d&#39;automatiser la caractérisation et le suivi de la performance cytomètre en utilisant les instructions du fabricant. </li><li> Quittez l&#39;application CS &amp; T. Appliquer paramètre CS &amp; T, assurez-vous que les paramètres de flux permettent encore un motif de gouttelettes breakoff stable, et tourner sur le bouton Sweet Spot comme décrit dans les instructions du fabricant (dans la fenêtre &quot;sécable&quot;). </li><li> Optimiser la valeur de retard Goutte utilisant des billes fluorescentes (voir le tableau des matériaux). <ol><li> Installez les tubes de prélèvement dans le support. Ouvrez la porte du bloc sort. Dans la fenêtre &quot;Side Stream&quot;, allumer la tension puis sélectionnez &quot;Sorte de test &quot;et cliquez sur le bouton Aspirateur tiroir pour ouvrir le tiroir et avoir accès à des tubes de prélèvement. </li><li> Optimiser les flux secondaires alors ils vont dans le centre du tube. Réglez les valeurs et les curseurs au besoin. </li><li> Fermez la porte du bloc de tri et assurez-vous de régler la molette de micromètre pour obtenir le point le plus lumineux de perles au centre. Tiroir à déchets Désélectionner, trier et la tension test. Retirer les tubes de prélèvement. </li><li> Optimiser le retard de chute selon les instructions du fabricant. <ol><li> En bref, secouer vigoureusement cordon flacon et diluer 1 goutte de perles dans 0,5 ml de PBS. </li><li> En utilisant les instructions du fabricant, définir le délai de dépôt en utilisant le modèle de l&#39;expérience Retard baisse disponible dans le logiciel. Vous pouvez également utiliser la fonction Auto Delay déplacer pour définir le délai de baisse. </li><li> Chargez le tube de perles fluorescentes et ajuster le débit jusqu&#39;à ce que le taux de seuil est ~ 3.000 à 4.000 événements / seconde. Réglez la précision de sorte à &quot;initiale&quot; et finaleLy à «peaufiner» dans la fenêtre «Trier de mise en page&quot;. Sélectionnez le filtre optique et de tri des perles. Régler le retard Dérouler jusqu&#39;à ~ 100% sont classifiées vers la gauche. Remarque: Cette étape est essentielle pour faire en sorte que les cellules d&#39;intérêt sorte dans un courant latéral. Les billes et le filtre optique sont utilisés pour obtenir un écart proche de gouttelette de 100%. </li></ol></li></ol></li><li> Placez un ND (densité neutre) filtre FSC 1.5 à l&#39;extrémité gauche du bloc de détecteur de FSC. Réglez l&#39;extension de la fenêtre à 2.00 ms et la mise à l&#39;échelle FSC Zone à 1,00 (dans l&#39;onglet &quot;laser&quot; dans la fenêtre &quot;cytomètre&quot;). </li><li> Avant de charger le tube de l&#39;échantillon, placer une ligne de filtration de l&#39;échantillon de 50 um à la fin de la ligne de prélèvement pour éviter l&#39;agglutination. Pour ce faire, sélectionnez &quot;Changer le filtre échantillon» dans le menu «cytomètre&quot;. </li><li> Testez la composition de l&#39;échantillon de fluide pour obtenir une bonne séparation sans attiser entre les flux de côté et d&#39;empêcher les cellules de coller à la polypropètubes NE. <ol><li> Chargez le tube d&#39;échantillon, tourner sur les plaques de déviation et sélectionnez &quot;sorte de test&quot; à tiroir fermé. Regardez les flux secondaires si la séparation se produit sans trop de Fanning. Remarque: Fanning est probablement due à une concentration élevée de protéine dans le tampon d&#39;échantillon. </li></ol></li><li> Chargez le tube d&#39;échantillon dans l&#39;instrument FACS. Sélectionner un échantillon agitation de 300 tours par minute et à une température de 4 ° C de l&#39;échantillon. Sélectionnez &quot;Acquérir les données&quot; dans le &quot;Tableau de bord de l&#39;acquisition». </li><li> Si nécessaire, diluer l&#39;échantillon d&#39;atteindre un taux d&#39;événement approprié pour un maximum de 5000 événements / sec (à un débit maximal de 3,0). Respecter strictement ces limites de façon à maintenir la pureté des cellules triées. </li><li> Pour analyser et trier les cellules, d&#39;optimiser la valeur de seuil de FSC pour éliminer les débris sans interférer avec la population d&#39;intérêt. Dans échelle logarithmique, ajuster la tension du détecteur tubes photo-multiplicateurs (PMT) avec le filtre de 530/30 afin de faireen sorte que le signal de fluorescence de la population totale à l&#39;intérieur de l&#39;échelle correspond, et porte sur la population positive (Figure 1, la porte 1). </li><li> Produire un Forward Scatter-zone (FSC-A) / Side Scatter-zone (SSC-A) parcelle de la population positive SYTO16 et ajuster la FSC-A à environ 110 V sur une échelle linéaire, et le SSC-A à environ 150 V en échelle logarithmique de visualiser tous les événements tout en excluant de très grandes cellules et les agrégats cellulaires. </li><li> Dans la fenêtre «Trier Layout&quot;, réglez le &quot;Mode Tri précision&quot; à &quot;la pureté 4-way&quot; (masque de Rendement: 0, Pureté Masque: 32, Phase Mask: 0). Sélectionnez «Continu» dans le menu «Événements cibles&quot; pour le tri continue. </li><li> Ajouter populations à trier dans le domaine correspondant aux tubes (dans la fenêtre &quot;Trier Layout&quot;). La place populations avec des fréquences plus élevées dans le milieu et ceux avec des fréquences inférieures aux extrémités. Réglez les plaques de tension à 2.500 V. Pendant le tri, garder le prélèvement de l&#39;échantillon tUBE sur la glace. </li></ol> 4. Tri cellulaire <ol><li> Immédiatement avant le tri, les cellules de filtrage utilisant un filtre de 50 um dans un 5 ml précédemment enduite polypropylène tube rond en bas. </li><li> Laver le filtre abondamment avec 1-2 ml de tampon de tri. </li><li> Cellules germinales Trier fixe utilisant un trieur de cellules laser équipé 488 nm suivant le schéma de déclenchement détaillé dans la figure 1. Recueillir des fractions purifiées dans 5 ml tubes en polypropylène à fond rond préalablement enduits à l&#39;article 1 sur la glace. <ol><li> Assurez-vous que 100-200 pi de FBS est maintenue dans les tubes de prélèvement pour éviter le collage des cellules triées à la paroi du tube. </li><li> Assurez-vous que la concentration de SYTO16 sature pour éviter les fluctuations de fluorescence au cours de la période de tri. SYTO16 sature lorsqu&#39;il n&#39;y a pas de variation en outre intensité de coloration des cellules à l&#39;Alexa Fluor 488 paramètre (coloration de l&#39;ADN) dans les graphiques de tri. Si nécessaire, ajouter 1 pl deSYTO16 au tube de tri pour atteindre la saturation. </li><li> Afficher les événements au total sur un graphique affichant les paramètres suivants: nombre d&#39;événements vs Alexa Fluor 488-A. </li><li> Sélectionner les cellules à coloration positive de l&#39;ADN avec une porte, comme représenté sur la figure 1. <ol><li> Afficher les cellules de la porte du 1 sur un graphique en utilisant SSC-A vs FSC-A en tant que paramètres <ol><li> Sélectionnez les cellules avec Gate 2, comme le montre la figure 1. </li><li> Afficher les cellules de Gate 2 sur un graphique en utilisant FSC-A vs Alexa Fluor 488-A en tant que paramètres. </li><li> Sélectionnez les cellules avec Porte 5 et 6 porte sur Gate 2 graphique, comme le montre la figure 1. </li><li> Afficher séparément Porte 5 et 6 porte sur des graphiques en utilisant Alexa Fluor 488-W vs Alexa Fluor 488-A en tant que paramètres. </li><li> Sélectionnez spermiogenèse étapes 1-9 spermatides sur le graphique Porte 5 et spermiogenèse étapes 10-12 spermatides sur la grille 6 graphiques, comme le montre la figure 1. </li></ol></li><li> Affichageles cellules de la porte du 1 sur un graphique en utilisant FSC-A vs Alexa Fluor 488-A en tant que paramètres <ol><li> Sélectionnez les cellules avec les entrées 3 et 4, comme le montre la figure 1. </li><li> Afficher séparément Porte 3 et la porte 4 graphiques utilisant Alexa Fluor 488-W vs Alexa Fluor 488-A en tant que paramètres. </li><li> Sélectionnez spermiogenèse étapes 13-14 spermatides sur le graphique Porte 3 et spermiogenèse étapes 15-16 spermatides sur le graphique porte 4, comme le montre la figure 1. </li></ol></li></ol></li></ol></li></ol>

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The authors declare no competing financial interests.

Les cellules de la spermatogenèse ont toujours été difficiles à étudier étant donné la complexité de l&#39;épithélium séminifère, ainsi que le succès limité de la culture in vitro. Au fil des ans, de nombreuses approches pour purifier les cellules germinales à partir de diverses espèces ont été développés. En utilisant des techniques de purification sédimentation par gravité avec des gradients de Percoll ou d&#39;albumine bovine de sérum fournissent un bon rendement des cellules germinale...

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 différenciation des spermatides de souris est un processus critique pour la production d&#39;un gamète mâle fonctionnel avec un génome intact à être transmis à la génération suivante. Jusqu&#39;à présent, les études moléculaires de cette transition morphologique ont été entravés par l&#39;absence d&#39;une méthode permettant une séparation adéquate de ces étapes importantes de la différenciation des spermatides pour des analyses ultérieures. Les tentatives précédentes pour bon déclenchement de ces cellules en utilisant la cytométrie de flux ont peut-être été difficile en raison d&#39;une augmentation particulière de la fluorescence de l&#39;ADN dans les spermatides subissant remodelage de la chromatine. Partant de ce constat, nous fournissons des détails d&#39;un simple, cytométrie de flux système, permettant la purification reproductible de quatre populations de spermatides de souris fixes avec de l&#39;éthanol, représentant chacun un état différent dans le processus de remodelage nucléaire. L&#39;enrichissement de la population est confirmée en utilisant des marqueurs spécifiques étapes et critères morphologiques. Les spermatides purifiés peuvent être utilisés pour la génomique et de protéomiqueic analyse.

Stratégie d&#39;entrée sont utilisés avec la cytométrie en flux Figure 1 représente la stratégie de déclenchement utilisé dans la cytométrie en flux pour trier quatre populations de spermatides très pures. Brièvement, les cellules dont la coloration de l&#39;ADN positif (Alexa Fluor 488-A) sont d&#39;abord sélectionné avec la Porte 1. Spermatides de spermiogenèse steps 1-12 sont sélectionnés (G...

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.

-Étape spécifique de tri Spermatides souris par cytométrie en flux

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

10.5K vues.  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.

Vidéo: 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.

Identification des progéniteurs érythromyélidiens et de leur descendance dans l&#39;embryon de souris par cytométrie de flux

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

Recherche

Biologie du développement

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.

Isoler et analyse des cellules du pancréas Mesenchyme par cytométrie de flux

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.

Une approche standardisée pour la purification multispécifique des cellules germinales masculines de mammifères par dissociation mécanique des tissus et cytométrie de flux

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.

Utilisation de la greffe de cellules souches hématopoïétiques pour évaluer l’origine du Syndrome myélodysplasique

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.

Cytométrie en cellules vivantes en temps réel d’enquêter sur l’Influx calcique, la Formation de pores et phagocytose par les récepteurs P2X7 dans des cellules progénitrices neurales adultes

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.

Isolement et coloration des kératinocytes de peau de souris pour l'analyse spécifique de cycle cellulaire de l'expression cellulaire de protéine par cytométrie de masse

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.

Enrichissement des spermatozoïdes pachytenés et des spermatides à partir de testeurs de souris à l'aide d'équipement de laboratoire standard

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.

Utilisation de la cytométrie du débit pour détecter et quantifier la formation de sang altérée chez le poisson zèbre en développement

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-isolement et culture de progéniteurs fibro-adipogènes et de cellules souches musculaires à partir de muscles squelettiques de souris non perturbés et blessés

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.

Identification et isolement de progéniteurs érythroïdes unitaires formant des éclatements et unités formant colonies à partir de tissus murins par cytométrie en flux

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