Gli autori desiderano ringraziare il Dr. Leonid Volkov e Éric Bouchard per la loro consulenza tecnica in materia di epifluorescenza. Sostegno finanziario Finanziato dal Canadian Institutes of Health Research (sovvenzione # MOP-93.781) a GB

Cura degli animali era in conformità con la cura degli animali e l&#39;uso comitato Université de Sherbrooke. 1. Preparazione del tubo <ol><li> Il giorno prima separazione delle cellule, aggiungere 1-2 ml di siero inattivato al calore fetale bovino (FBS) a 5 ml in polipropilene provette con fondo arrotondato, e di 15 ml e 50 ml tubi in polipropilene coniche. Passaggio fondamentale: Assicurarsi che ogni tubo utilizzato nel protocollo è rivestito. Nota: Rivestimento FBS impedisce alle cellule germinali di attaccarsi alle pareti del tubo. </li><li> Mescolare lentamente O / N per inversione a 4 ° C per rivestire i tubi in modo uniforme con un rotatore. </li><li> Il giorno successivo, togliere FBS da tubi rivestiti per decantazione. <ol><li> * Passo critico: Assicurarsi che i 5 ml in polipropilene provette con fondo arrotondato e le 15 e 50 ml tubi utilizzati per la preparazione delle cellule (protocollo passaggi 2,1-2,11) sono completamente svuotati di FBS per prevenire Fanning (ad esempio, deviazione imprecisa dei torrenti laterali). Utilizzare una pipetta P200 per rimuovere le FBS. <br/&gt; Nota: residua FBS in tubi prima di smistamento può portare a una perdita di cellule o la contaminazione di altri tubi utilizzati durante l&#39;ordinamento delle cellule, </li><li> Lascia un piccolo volume residuo di FBS (circa 100-200 ml) in polipropilene da 5 ml tubi tondi fondo utilizzati per la raccolta delle cellule purificate. </li></ol></li></ol> 2. Preparazione delle cellule <ol><li> Sacrifica un topo maschio in anestesia con O 2 / isoflurano (induzione al 5% poi mantenuto al 2%), seguito da CO 2 asfissia. </li><li> Accise e decapsulate entrambi i testicoli e tritare con piccole forbici in 500 ml di smistamento tampone in una provetta da 1,5 ml. </li><li> Cellule germinali Flush da tubuli seminiferi di dolce pipettando su e giù nel tubo 1,5 ml, prima con una punta tronca 100-1000 ml, e poi con un intatto punta 100-1000 ml. </li><li> Rimuovere i detriti e ciuffi dopo passo una filtrazione con un colino cella 40 micron e raccogliere filtrato nei 50 ml prev tubo conicoiously rivestito con FBS al punto 1. </li><li> Lavare il filtro una volta con tampone di smistamento fino ad un volume totale di 3 ml e trasferimento filtrato nel tubo da 15 ml precedentemente rivestito in passaggio 1. </li><li> Aggiungere 16 ml di 50 mM EDTA pH 8 per millilitro di sospensione cellulare (48 ml per 3 ml) in modo da ottenere una concentrazione finale di 0,8 mM EDTA. </li><li> Per risolvere le cellule germinali, lentamente aggiungere 3 volumi di ghiacciata etanolo al 100% con un 10 ml pipetta con agitazione (vortex a bassa velocità), che produrrà una sospensione lattiginosa. Step critico: lentamente aggiungendo etanolo è cruciale e importante preservare l&#39;integrità della preparazione cellulare. Abbiamo notato che la rapida aggiunta di etanolo provoca la lisi delle cellule con una conseguente notevole diminuzione dell&#39;efficienza della selezione. Per una sospensione cellulare 3 ml, 9 ml di etanolo dovrebbe essere aggiunto in circa 1 min. </li><li> Incubare le cellule in ghiaccio per 15 minuti con miscelazione occasionali per inversione. </li><li> Sospensione cellulare a 800 xg Centrifugare per 5 min e rimuovere il chiarosurnatante. </li><li> Risospendere delicatamente le cellule germinali fissati in 2 ml di cernita tampone. </li><li> Aggiungere 4 ml di colorante DNA SYTO16 (soluzione 1 mM in DMSO) e incubare per almeno 30 min (protetto dalla luce). Nota: I risultati migliori si ottengono utilizzando ordinamento SYTO16 poiché è un colorante DNA permeabile che è facilmente incorporato in nuclei altamente compattati di spermatidi. </li></ol> Sorter 3. cellulare Set Up Viene utilizzato 20 parametri cell sorter BDFACSAria III Ecco, un 4-Laser (rosso 405 nm - viola, 488 nm - blu, 561 nm - - giallo-verde, 633 nm): Nota. Il software BD FACSDiva 6.1.3 viene utilizzato per visualizzare e analizzare i dati. Le impostazioni possono variare a seconda del tipo di sorter utilizzato. <ol><li> Sterilizzare il sorter FACS eseguendo il 70% di etanolo come fluido di trasporto durante la procedura di spegnimento fluidica (menu &quot;Cytometer&quot; del software) prima di smistamento. Selezionare &quot;Citometro&quot; nella barra degli strumenti del software e scegliere &quot;sh Fluidicutdown &quot;. Seguire la procedura citati dai processi. </li><li> Preparare e filtrata 1x PBS con un filtro da 0,22 micron in modo da eliminare eventuali cristalli e contaminanti. Riempire il serbatoio guaina la linea di saldatura superiore all&#39;interno del serbatoio. </li><li> Avviare il software ed eseguire il login. Avviare il sorter FACS a scaldare i laser per almeno 30 minuti prima di smistamento. Eseguire due processi di avvio fluidici per scovare l&#39;etanolo dai fluidica. Selezionare &quot;Citometro&quot; nella barra degli strumenti del software e scegliere &quot;avvio Fluidic&quot;. Seguire i passaggi indicati dai processi. Nota: Questo ripristina fluidica con normale liquido guaina (1x PBS). </li><li> Pulire il blocco di ordinamento e le piastre di deflessione con acqua distillata e asciugare accuratamente. Sonicare un ugello 100 micron posto in un tubo di acqua distillata per 2 minuti in un bagno di sonicazione. Pulire a fondo l&#39;ugello. </li><li> Inserire l&#39;ugello 100 micron nella cella di flusso e ruotare l&#39;ugello leva di bloccaggio in senso orario al 12Posizione hr. Impostare la pressione della guaina di 20 psi. </li><li> Accendere il torrente e regolare l&#39;ampiezza di ottenere valori di riferimento per la frequenza (circa 30), Goccia 1 (circa 150) e Gap (circa 12), e di stabilire un modello di gocce di troncaggio stabile (gocce pochi satellite). Spegnere attenuazione. </li><li> Assicurarsi che il flusso sia diritto e colpisce il centro dell&#39;aspiratore rifiuti cambiando l&#39;angolo del blocco genere. Nota: Un ugello 130 micron a 10 psi è stato anche testato ed è stato trovato nessuna differenza evidente. </li><li> Impostare ritardo laser a 0 per il blu, -77,39 per il rosso, 37.33 per il viola e -39,85 per i laser di colore giallo-verde. </li><li> Impostazione delle aree di scala di 1,14 per il blu, 1.0 per il rosso, 0,75 per la viola e 0.96 per i laser di colore giallo-verde. </li><li> Utilizzando un Sort Test (nella finestra &quot;laterale Stream&quot; del software), fare in modo che i flussi laterali non sono Fanning. Nella finestra &quot;laterale Stream&quot;, regolare i cursori di tensione di ogni flusso lato in modo che hanno colpito il middle di tubo di raccolta di prova. Se il flusso centrale non è stretto, regolare il 2 °, 3 ° e 4 impostazioni ° drop per limitare il flusso centrale. </li><li> Avviare l&#39;installazione citometro e tracking (CS &amp; T) applicazione nel (menu &quot;Cytometer&quot;) del software. </li><li> Utilizzare l&#39;Installazione e monitoraggio perline citometro a 1 goccia per 350 ml per automatizzare la caratterizzazione e il monitoraggio delle prestazioni citometro utilizzando le istruzioni del produttore. </li><li> Chiudere l&#39;applicazione CS &amp; T. Applicare impostazione CS &amp; T, assicurarsi che i parametri di flusso consentono ancora un andamento stabile delle gocce di troncaggio, e attivare il pulsante di Sweet Spot come descritto nelle istruzioni del produttore (nella &quot;finestra breakoff&quot;). </li><li> Ottimizzare il valore di goccia di ritardo utilizzando perline fluorescenti (vedi Tabella dei Materiali). <ol><li> Installare i tubi di raccolta nel supporto. Aprire la porta blocco tipo. Nella finestra &quot;laterale Stream&quot;, accendere la tensione, quindi selezionare &quot;Test di sorta &quot;e fare clic sul pulsante aspiratore cassetto per aprire il cassetto e avere accesso ai tubi di raccolta. </li><li> Ottimizzare i flussi laterali così vanno al centro del tubo. Regolare i valori e cursori come necessario. </li><li> Chiudere la porta blocco tipo e assicurarsi di regolare il quadrante del micrometro per ottenere il posto tallone luminoso al centro. Cassetto rifiuti deselezionare, prova sorta e la tensione. Rimuovere i tubi di raccolta. </li><li> Ottimizzare il ritardo calo secondo le istruzioni del fabbricante. <ol><li> In breve, agitare tallone fiala vigorosamente e diluire 1 goccia di perline in 0,5 ml di PBS. </li><li> Utilizzando le istruzioni del produttore, impostare il ritardo goccia utilizzando il modello esperimento goccia Ritardo disponibili nel software. In alternativa, utilizzare la funzione Auto Goccia Ritardo per impostare il ritardo goccia. </li><li> Caricare il tubo di perline fluorescenti e regolare la portata fino a quando il tasso di soglia è ~ 3.000 a 4.000 eventi / secondo. Impostare la precisione sorta di &quot;iniziale&quot; e finalemente a &quot;sintonizzare&quot; nella finestra &quot;Layout Sort&quot;. Selezionare il filtro ottico e ordinare perline. Regolare il ritardo di goccia fino ~ 100% sono allineati a sinistra. Nota: Questo passaggio è fondamentale per fare in modo che le cellule di interesse specie in un flusso laterale. Le perline e il filtro ottico sono utilizzati per ottenere vicino a una deviazione gocciolina 100%. </li></ol></li></ol></li><li> Posizionare un ND (neutral density) Filtro FSC 1.5 all&#39;estremità sinistra del blocco rilevatore FSC. Impostare l&#39;estensione della finestra di 2.00 ms e il ridimensionamento FSC area a 1,00 (nella scheda &quot;Laser&quot; nella finestra &quot;citometro&quot;). </li><li> Prima di caricare provetta posizionare una linea di filtro campione di 50 um alla fine della linea di campionamento per evitare grumi. Per farlo, selezionate &quot;Modifica filtro campione&quot; nel menu &quot;citometro&quot;. </li><li> Testare la composizione del fluido campione per ottenere una buona separazione senza ventaglio tra flusso laterale e per evitare che le cellule di attaccarsi alla poliprotubi ne. <ol><li> Caricare la provetta, accendere le piastre di deflessione e selezionare &quot;Test tipo&quot; con il cassetto chiuso. Guardate i flussi laterali in caso di separazione senza troppi Fanning. Nota: Fanning è probabilmente dovuto ad un alta concentrazione di proteine ​​nel tampone. </li></ol></li><li> Caricare la provetta nello strumento FACS. Selezionare un campione di agitazione 300 rpm ed una temperatura del campione di 4 ° C. Selezionare &quot;Acquisire dati&quot; nella &quot;Acquisizione Dashboard&quot;. </li><li> Se necessario, diluire il campione per ottenere un tasso di evento appropriato ad un massimo di 5000 eventi / sec (ad una portata massima di 3,0). Rigorosamente rispettare questi limiti in modo da mantenere la purezza delle cellule ordinati. </li><li> Per analizzare e ordinare le celle, ottimizzare il valore di soglia FSC per eliminare i residui senza interferire con la popolazione di interesse. In scala logaritmica, regolare la tensione del rivelatore con il filtro 530/30 tubo fotomoltiplicatore (PMT) per rendereAssicurarsi che il segnale di fluorescenza della popolazione totale si inserisce all&#39;interno della scala, e porta sulla popolazione positivo (Figura 1, Gate 1). </li><li> Produrre un Forward Scatter-zona (FSC-A) / Side Scatter-zona (SSC-A) diagramma della popolazione positivo SYTO16 e regolare la FSC-A a circa 110 V in scala lineare, e la SSC-A a circa 150 V in scala logaritmica per visualizzare tutti gli eventi escludendo molto grandi cellule e aggregati cellulari. </li><li> Nella finestra &quot;Ordina Layout&quot;, impostare il &quot;modo Ordinamento Precision&quot; a &quot;purezza 4-way&quot; (maschera Yield: 0, Purezza Maschera: 32, Fase Maschera: 0). Selezionare &quot;Continua&quot; dal menu &quot;Target Eventi&quot; per l&#39;ordinamento continuo. </li><li> Aggiungere popolazioni per ordinare nel campo corrispondente ai tubi (nella finestra &quot;Sort Layout&quot;). Popolazioni posto con frequenze più alte al centro e quelli con frequenze più basse alle estremità. Impostare le piastre di tensione a 2.500 V. Durante l&#39;ordinamento, mantenere la raccolta del campione tUBE su ghiaccio. </li></ol> 4. cella Ordinamento <ol><li> Immediatamente prima di smistamento, celle filtranti usando un filtro 50 micron in un precedentemente rivestito 5 ml in polipropilene tubo tondo fondo. </li><li> Lavare il filtro a lungo con 1-2 ml di cernita tampone. </li><li> Cellule germinali Ordina fissi utilizzando un attrezzato laser cell sorter 488 nm secondo lo schema di gating dettagliato in figura 1. Raccogliere frazioni purificate in polipropilene 5 ml provette con fondo arrotondato precedentemente rivestite nella sezione 1 su ghiaccio. <ol><li> Assicurarsi che 100-200 ml di FBS è conservato nei tubi di raccolta per evitare incollaggio delle cellule ordinati alla parete del tubo. </li><li> Assicurarsi che la concentrazione di SYTO16 è saturare per evitare fluttuazioni di fluorescenza durante il periodo di smistamento. SYTO16 è saturare quando non c&#39;è un&#39;ulteriore variazione di intensità di colorazione della cellula al 488 parametro Alexa Fluor (colorazione DNA) nella grafica smistamento. Se necessario, aggiungere 1 ml diSYTO16 al tubo di smistamento per raggiungere la saturazione. </li><li> Visualizzare gli eventi totali su un grafico che visualizza i seguenti parametri: numero di eventi vs Alexa Fluor 488-A. </li><li> Selezionare le celle colorazione DNA positivi con Gate 1, come mostrato in Figura 1. <ol><li> Celle di visualizzazione dalla porta 1 su un grafico con SSC-A vs FSC-A come parametri <ol><li> Selezionare le celle con Gate 2, come mostrato in Figura 1. </li><li> Celle di visualizzazione dalla Porta 2 su un grafico utilizzando FSC A vs Alexa Fluor 488-A come parametri. </li><li> Selezionare celle con Gate 5 e 6 su Gate Porta 2 grafica, come mostrato in Figura 1. </li><li> Visualizza separatamente Gate 5 e Gate 6 sulla grafica che utilizzano Alexa Fluor 488-W vs Alexa Fluor 488-A come parametri. </li><li> Selezionare spermiogenesi i passaggi 1-9 spermatidi sul Gate 5 grafica e spermiogenesis passaggi 10-12 spermatidi sul cancello 6 grafici, come mostrato in Figura 1. </li></ol></li><li> Displaycellule dalla porta 1 su un grafico utilizzando FSC A vs Alexa Fluor 488-A come parametri <ol><li> Selezionare le celle con Gate 3 e Gate 4, come mostrato in Figura 1. </li><li> Visualizza separatamente Gate 3 e Gate 4 sulla grafica che utilizzano Alexa Fluor 488-W vs Alexa Fluor 488-A come parametri. </li><li> Selezionare spermiogenesi passaggi 13-14 spermatidi sul Gate 3 grafica e spermiogenesis passaggi 15-16 spermatidi sulla Porta 4 grafico, come mostrato in Figura 1. </li></ol></li></ol></li></ol></li></ol>

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The authors declare no competing financial interests.

Cellule spermatogenic sono sempre stati difficili da studiare data la complessità del seminifero, nonché il limitato successo della coltura in vitro. Nel corso degli anni, molti approcci per purificare cellule germinali di varie specie sono stati sviluppati. Tecniche di sedimentazione che utilizzano purificazione gravità con Percoll o bovina siero gradienti di albumina solitamente forniscono una buona resa di cellule germinali intatte, ma mancano di adeguata definizione tra alcuni tipi di cellule come le cel...

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 differenziazione di spermatidi topo è uno processo critico per la produzione di un gamete maschile funzionale con un genoma intatto da trasmettere alla generazione successiva. Finora, gli studi molecolari di questa transizione morfologica sono stati ostacolati dalla mancanza di un metodo che consente un&#39;adeguata separazione di queste importanti fasi di differenziazione spermatid per analisi successive. Precedenti tentativi di gate adeguato di queste cellule in citometria a flusso potrebbe essere stato difficile a causa di un particolare aumento della fluorescenza del DNA in spermatidi in fase di rimodellamento della cromatina. Sulla base di questa osservazione, noi forniamo i dettagli di un semplice sistema di citometria a flusso, che permette la purificazione riproducibile di quattro popolazioni di spermatidi topo fissate con etanolo, ognuno dei quali rappresenta uno stato diverso nel processo di rimodellamento nucleare. L&#39;arricchimento della popolazione è confermato per mezzo di marcatori specifici per step e criteri morfologici. Gli spermatidi purificati possono essere utilizzati per genomica e proteomic analisi.

Strategia di gating utilizzato con citometria a flusso La figura 1 rappresenta la strategia di gating utilizzato in citometria a flusso per ordinare quattro popolazioni spermatidi altamente puri. In breve, le cellule con colorazione del DNA positivo (Alexa Fluor 488-A) sono selezionati prima con Porta 1. spermatidi da spermiogenesis Predellino 1-12 sono selezionati (Porta 2) su un diagramma a punti che mostra ...

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.

Specifiche Step-ordinamento di mouse spermatidi mediante citometria di flusso

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 Views.  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.

Identificazione di progenitori eritromieloidi e loro progenie nell&#39;embrione del mouse dalla citometria del flusso

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

Ricerca

Biologia dello sviluppo

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.

Isolare e analisi cellule del pancreas Mesenchima mediante citometria di flusso

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 approccio standardizzato per la depurazione multispecie di cellule germinali maschili di mammiferi mediante dissociazione tissutale meccanica e citometria del flusso

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 del trapianto di cellule staminali ematopoietiche per valutare l'origine della sindrome Myelodysplastic

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.

In tempo reale Live-cella flusso Cytometry per indagare l'afflusso del calcio, la formazione del poro e fagocitosi dai recettori P2X7 in cellule progenitrici neurali adulto

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.

Isolamento e colorazione dei Keratinociti della pelle del topo per l'analisi specifica del ciclo cellulare dell'espressione proteica cellulare da citometria di massa

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.

Arricchimento di spermatociti pachytene e Spermatidi da test del mouse utilizzando attrezzature di laboratorio 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.

Utilizzo della citometria a flusso per rilevare e quantificare la formazione di sangue alterato nel pesce zebra in via di sviluppo

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-isolamento e coltura di progenitori fibro-adipogeni e cellule staminali muscolari da muscolo scheletrico di topo imperturbato e ferito

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.

Identificazione e isolamento di progenitori eritroidi dell'unità burst-forming e dell'unità formante colonie da tessuto murino mediante citometria a flusso

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