Questo progetto è stato sostenuto in parte dal denaro dallo Stato della Florida a Scripps e numeri premio R01GM085079 e R21HD061304 dal National Institute of General Medical Sciences e il National Institute of Child Health e lo Sviluppo Umano, rispettivamente. Questo è il numero manoscritto 20917 di The Scripps Research Institute.

Questo protocollo può essere diviso in due fasi principali: (1) la colorazione 33342 dissociazione e Hoechst di mouse cellule testicolo seguito, se necessario, (2) FACS ordinamento delle frazioni rilevanti meiotico, comprese tutte le fasi della meiosi, da cellule germinali di spermatidi rotondi. Una volta raccolte, queste popolazioni altamente arricchito meiotica può essere utilizzato per una vasta gamma di analisi. Questo protocollo descrive la dissociazione di un testicolo adulto; volumi possono essere adattate di conseguenza per i minori o per testicoli aggiuntivi. 1. Testicolo dissociazione <ol><li> Mettete un testicolo decapsulated in un tubo da 15 ml. </li><li> Aggiungere 3 ml di soluzione Balance Gey di Salt (GBSS) contenente 120 U / ml di Collagenasi tipo I. </li><li> Aggiungere 10 ml di DNAsi I (soluzione stock 1mg/ml nel 50% glicerolo) e scuoterlo energicamente a mano fino a quando vedrete tubuli testicolari iniziando a dissociarsi. </li><li> Agitare orizzontalmente ad un massimo di 120 rpm per 15 minuti a 33 ° C. </li><li> Decantare per 1 minuto in verticale a temperatura ambiente e scartare il surnatante. </li><li> Ripetere i passaggi 1,2-1,5. </li><li> Aggiungere 2,5 ml di GBSS contenente Collagenasi di tipo I, 50 microlitri di una soluzione madre 50mg/ml tripsina risospese in soluzione 1mM HCl, e 10 ml di DNAsi I (1mg/ml), e capovolgere il tubo più volte. </li><li> Agitare orizzontalmente ad un massimo di 120 rpm per 15 minuti a 33 ° C. </li><li> Utilizzando plastica pipetta Pasteur monouso con orifizio largo, pipettare delicatamente su e giù per 3 grumi min.No dovrebbe essere visibile a questo punto. </li><li> Aggiungere 30 ml di tripsina, 10 ml di DNAsi I, 40 ml di Hoechst 33342 risospeso in DMSO (10 mg / ml), e capovolgere il tubo più volte. </li><li> Agitare orizzontalmente ad un massimo di 120 rpm per 15 minuti a 33 ° C. </li><li> Aggiungere 400 ml di siero fetale bovino (FCS) e mescolare invertendo per inattivare la tripsina. </li><li> Colorazione finale è eseguita aggiungendo 50 l di Hoechst 33342 risospeso in DMSO (10 mg / ml) e 10 ml di DNAsi I (1 mg / ml). </li><li> Agitare orizzontalmente ad un massimo di 120 rpm per 15 minuti a 33 ° C. </li><li> Il campione testicolo dissociata viene fatto passare attraverso due GBSS 40-micron filtri usa e getta di pre-contatto con il fluido in un tubo da 50 ml conica, poi 5 ml di ioduro di propidio (PI) si aggiunge la soluzione e il campione viene delicatamente mescolato pipettando più volte con Pasteur monouso pipetta. </li><li> Campione viene trasferito in una siringa da 5 ml in plastica attraverso un ago calibro 18. Quest&#39;ultima viene sostituita da una 22-gauge per la consegna del campione. La siringa è memorizzato sul ghiaccio e al riparo dalla luce fino al momento per l&#39;elaborazione FACS. </li></ol> 2. FACS installazione e purificazione <ol><li> L&#39;ordinamento è stato eseguito su un Becton Dickinson-cell sorter Aria IIU. Le condizioni descritte dovrebbero pertanto essere utilizzate come punto di partenza soprattutto quando si utilizzano apparecchiature differenti. Abbiamo usato condizioni precedentemente descritte 7,8. </li><li> Dato che la IIU Aria non ha un laser UV, abbiamo adattato il protocollo per la rilevazione di colorazione Hoescht utilizzando un laser 405nm viola in aggiunta abbiamo utilizzato un laser blu 488 nm per il rilevamento scatter in avanti e laterale. Inoltre, alcuni filtri sono stati modificati al fine di limitare alcune permeabilità laser rosso con conseguente miglioramento della nitidezza trama sparsi. </li><li> Il laser viola è stato configurato con una banda passante 450/40nm per il rilevamento delle emissioni Hoescht blu e una banda passante 585/42nm per l&#39;emissione Hoescht Rosso. Un passaggio 502nm lungo è stata utilizzata per separare blu fluorescenza rossa. </li><li> A 100 micron ugello è stata utilizzata con una frequenza goccia disco di 28.000 gocce / secondo. Il tasso soglia campione è stato di circa 4000 eventi / secondo. L&#39;opzione di controllo della temperatura è stata utilizzata per mantenere campioni e provette a 4 ° C tutta la durata della selezione. Inoltre, la funzione agitazione campione è stato utilizzato a 200 giri al minuto per evitare che il campione dalla sedimentazione nel corso del genere. </li><li> Di solito, due o tre testicoli sono stati elaborati per 6 specie ora che permette la raccolta di 0.5-2.0x10 6 celle per ogni popolazione (vedere la sezione rappresentante). </li><li> Il campione è stato ordinato in aliquote di circa 750μl dispensato dalla siringa. Nel frattempo, durante queste pause le provette di raccolta sono stati mantenuti a 4 ° C, al riparo dalla luce, e delicatamente miscelati prima di ordinare la ripresa. </li><li> I campioni raccolti sono stati ordinati in una 12x75mm tubo di raccolta di vetro borosilicato contenente 250μl DMEM supplementato con 10% FCS. </li></ol> 3. Rappresentante dei risultati: Un profilo tipico FACS è mostrata in Figura 1. Tutte le fasi principali della meiosi sono identificati e sono indicati in legenda. Se l&#39;Hoechst 33342 macchia non è ottimale, il profilo globale appare molto più compatto e meno definite. Un problema comune riguarda la non sufficiente l&#39;aggiunta di DNAsi, che inducono una rapida aggregazione cellulare. Aggiunta di olio extra DNAsi a tutti i passaggi incriminati risolve di solito questo problema. Congelare °awing dello stock DNAsi (conservati a -20 ° C) dovrebbe essere ridotto al minimo, come questo si traduce in una rapida perdita di attività enzimatica. Spo11 è la endonucleasi meiotica che dirige rotture del doppio filamento in siti di hotspot ricombinazione meiotica 12. La figura 2 mostra i profili tipici di un Spo11 nullo testicolo dove doppio filamento pausa non si formano, con un conseguente meiosi abortivo 12, così come un pre-puberous profilo di un 13-giorni vecchio mouse, che rivela la natura asincrona di questa prima ondata di meiosi. Standard di analisi citogenetica utilizzando la combinazione di proteine ​​complesse sinaptonemalico 3 (SCP3) e fosforilato anticorpi H2AX fase-specifici marcatori meiosi dovrebbe essere inizialmente utilizzato per confermare la purezza e la natura delle cellule ordinati meiotico, come descritto jazz band 11. <img src="/files/ftp_upload/2602/2602fig1.jpg" alt="Figura 1" /> Figura 1. Wild-type FACS meiosi profili. A) scatter plot Rappresentante è mostrato. Le cellule gated sono mostrati. Notare la grande quantità di detriti presenti nel testicolo adulto contenente per lo più vuote e le membrane code spermatidi. B) Una trama rappresentante PI mostra la quantità molto limitata di cellule PI positivi presenti in un campione. Il cancello tipico è mostrato. C) Un rappresentante wild-type Hoechst 33342 profilo FACS è mostrato. Le varie popolazioni di cellule meiotica che può essere purificato usare questo metodo per ulteriori studi sono indicati: spermatogoni (Sp), pre-leptotene (PL), leptotene-zigotene (L / Z), pachitene precoce (EP), medio-pachitene ( spermatidi mP), tardo-pachitene (LP), diplotene (D), e tonde (RS). <img src="/files/ftp_upload/2602/2602fig2.jpg" alt="Figura 2" /> Figura 2. Spo11 nullo e wild-type giorno 13 profili meiotica FACS. A) Un tipico Spo11-null profilo viene mostrato con un chiaro fallimento meiotica, dove nessuna fase passato leptotene / zigotene precoce pachitene possono essere rilevati. B) Il profilo di un 13-giorno-vecchio wild-type topo maschio dimostra l&#39;alta concentrazione di cellule leptotene / zigotene. Questa prima ondata di meiosi è piuttosto asincrono, come chiaramente visualizzate utilizzando questa metodologia.

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

Nessun conflitto di interessi dichiarati.

Il protocollo presentato qui permette di purificare contemporaneamente da topi maschi adulti l&#39;intera gamma delle cellule stadio meiotico con purezza molto elevata, permettendo agli investigatori di studiare la dinamica di questo processo fondamentale. Cellule purificate possono essere utilizzati per numerose applicazioni che vanno dalla RNA 9,10, la mappatura nucleosoma 11, il rilevamento molecola ricombinante, analisi delle proteine, e molti altri. Tuttavia, i metodi di rilevazione devono ess...

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

flow cytometry purification of mouse meiotic cells

La natura eterogenea di tipi cellulari nel testicolo e l&#39;assenza di modelli di coltura cellulare meiotico sono stati ostacoli significativi allo studio dei programmi di differenziazione unico che si manifestano durante la meiosi. Due metodi principali sono state sviluppate per purificare, a livelli diversi, diverse frazioni meiotica sia da adulti e animali immaturi: elutriation o Staput (sedimentazione) utilizzando la BSA e / o gradienti di Percoll. Entrambi questi metodi si basano sulla dimensione della cella e la densità di separare le cellule meiotico 1-5. Nel complesso, ad eccezione di popolazioni cellulari pochi 6, questi protocolli non riescono a produrre una sufficiente purezza delle numerose popolazioni di cellule meiotica che sono necessari per la dettagliata analisi molecolari. Inoltre, con questi metodi di solito un tipo di cellule meiotico può essere purificato in un dato momento, che aggiunge un ulteriore livello di complessità quanto concerne la riproducibilità e omogeneità nel confronto tra campioni di cellule meiotica. Qui, descriviamo un metodo raffinato che permette di visualizzare facilmente, identificare e purificare cellule meiotico, da cellule germinali di spermatidi rotondi, usando FACS combinato con Hoechst 33342 colorazione 7,8. Questo metodo fornisce una fotografia complessiva dell&#39;intero processo meiotico e permette di purificare cellule vitali altamente dalla maggior parte fase della meiosi. Queste cellule purificate possono poi essere analizzati in dettaglio i cambiamenti molecolari che accompagnano la progressione attraverso la meiosi, per esempio le modifiche nell&#39;espressione genica 9,10 e le dinamiche di occupazione nucleosoma presso gli hotspot di ricombinazione meiotica 11.

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

Flow Cytometry Purification of Mouse Meiotic Cells

Un metodo efficace per ottenere altamente purificato frazioni praticabile meiotica da mouse testicolo è descritto, che unisce un raffinato cellulare dissociazione protocollo con fluorescenti separazione delle cellule attivate (FACS). Questo metodo sfrutta le differenze nel contenuto di DNA nucleare e la densità di discreta frazioni meiotico.

Purificazione Citometria a flusso di cellule di topo Meiotic

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

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

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Biology

Unit 1

Meiosis

SCIENTISTS IN ACTION

17.6K Views.  The Scripps Research Institute. Un metodo efficace per ottenere altamente purificato frazioni praticabile meiotica da mouse testicolo è descritto, che unisce un raffinato cellulare dissociazione protocollo con fluorescenti separazione delle cellule attivate (FACS). Questo metodo sfrutta le differenze nel contenuto di DNA nucleare e la densità di discreta frazioni meiotico.

Video: Flow Cytometry Purification of Mouse Meiotic Cells

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

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

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

La colorazione delle proteine ​​in gel con Coomassie G-250, senza solventi organici acido acetico e

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

Ricerca

Biologia

Flow Cytometry-based Purification of S. cerevisiae Zygotes

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

Flow Cytometry-based Purification of S. cerevisiae Zygotes

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

Flusso Cytometry basata Purificazione<em&gt; S. cerevisiae</em&gt; Zigoti

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

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

Ottimizzato Metodi di modellazione e colorazione proliferazione per il monitoraggio divisione cellulare utilizzando coloranti Tracking Cellulari

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

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

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

Misure calcio citosolico nelle cellule epiteliali renali mediante citometria di flusso

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

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon fertilization. The resulting aneuploidy in developing embryos is recognized as a major cause of pregnancy loss and congenital birth defects such as Down&#8217;s syndrome. Accurate chromosome segregation is critically dependent on the formation of the microtubule spindle apparatus, yet this process remains poorly understood in mammalian oocytes. Intriguingly, meiotic spindle assembly differs from mitosis and is regulated, at least in part, by unique microtubule organizing centers (MTOCs). Assessment of MTOC-associated proteins can provide valuable insight into the regulatory mechanisms that govern meiotic spindle formation and organization. Here, we describe methods to isolate mouse oocytes and deplete MTOC-associated proteins using a siRNA-mediated approach to test function. In addition, we describe oocyte fixation and immunofluorescence analysis conditions to evaluate meiotic spindle formation and organization.

Here, we present a protocol for specific siRNA-mediated mRNA depletion followed by immunofluorescence analysis to evaluate meiotic spindle assembly and organization in mouse oocytes. This protocol is suitable for in vitro depletion of transcripts and functional assessment of different spindle and/or MTOC-associated factors in oocytes.

Meiotic mandrino valutazione in mouse Ovociti mettendo a tacere siRNA-mediata

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon ...

LED Thermo Flow &#8212; Combining Optogenetics with Flow Cytometry

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the controlled delivery of light to the cell sample and hence the most popular tools for optogenetic studies are microscopy-based cell analyses and in vitro experiments. The flow cytometer has major advantages over a microscope, including the ability to rapidly measure thousands of cells at single cell resolution. However, it is not yet widely used in optogenetics. Here, we present a device that combines the power of optogenetics and flow cytometry: the LED Thermo Flow. This device illuminates cells at specific wavelengths, light intensities and temperatures during flow cytometric measurements. It can be built at low cost and be used with most common flow cytometers. To demonstrate its utility, we characterized the photoswitching kinetics of Dronpa proteins in vivo and in real time. This protocol can be adapted to almost all optically controlled substances and substantially expands the set of possible experiments. More importantly, it will greatly simplify the discovery and development of new optogenetic tools.

Optically controlled substances are powerful tools to study signaling pathways. To expand the spectrum of possible experiments, we developed a device for studying optically controlled substances in real time using flow cytometry: the LED Thermo Flow.

Thermo flusso LED - La combinazione Optogenetics con citometria a flusso

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the ...

Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes

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

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

Preparazione del cromosoma meiotico si diffonde da Mouse spermatociti

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

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

Analisi delle sospensioni cellulari isolate da tessuti solidi da Spectral citometria a flusso

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

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

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

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

Trans-inner Cell Mass iniezione delle cellule staminali embrionali porta a tassi più elevati di chimerismo

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

Measurement of Mitochondrial Mass and Membrane Potential in Hematopoietic Stem Cells and T-cells by Flow Cytometry

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production of all mature blood cells. In recent years cellular metabolism has emerged as a crucial regulator of HSC function and fate. We have previously demonstrated that modulation of mitochondrial metabolism influences HSC fate. Specifically, by chemically uncoupling the electron transport chain we were able to maintain HSC function in culture conditions that normally induce rapid differentiation. However, limiting HSC numbers often precludes the use of standard assays to measure HSC metabolism and therefore predict their function. Here, we report a simple flow cytometry assay that allows reliable measurement of mitochondrial membrane potential and mitochondrial mass in scarce cells such as HSCs. We discuss the isolation of HSCs from mouse bone marrow and measurement of mitochondrial mass and membrane potential post ex vivo culture. As an example, we show the modulation of these parameters in HSCs via treatment with a metabolic modulator. Moreover, we extend the application of this methodology on human peripheral blood-derived T cells and human tumor infiltrating lymphocytes (TILs), showing dramatic differences in their mitochondrial profiles, possibly reflecting different T cell functionality. We believe this assay can be employed in screenings to identify modulators of mitochondrial metabolism in various cell types in different contexts.

Here we describe a reliable method to measure mitochondrial mass and membrane potential in ex vivo cultured hematopoietic stem cells and T cells.

Misurazione della massa mitocondriale e del potenziale della membrana nelle cellule staminali ematopoietiche e t-cellule da parte della citometria di flusso

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production ...