Dieses Projekt wurde zum Teil durch Gelder aus dem Bundesstaat Florida, um Scripps und Vergabe Zahlen R01GM085079 und R21HD061304 vom National Institute of General Medical Sciences und dem National Institute of Child Health and Human Development, bzw. unterstützt. Dies ist Manuskript Nummer 20917 von The Scripps Research Institute.

Dieses Protokoll kann in zwei wesentliche Schritte unterteilt werden: (1) die Dissoziation und Hoechst 33342 Färbung der Maus Hoden-Zellen, gefolgt von, wenn nötig, (2) FACS-Sortierung der relevanten meiotischen Fraktionen in allen Phasen der Meiose, die aus Keimzellen zu runden Spermatiden. Einmal erfasst, können diese hoch angereichertes meiotischen Bevölkerung für eine breite Palette von Analysen verwendet werden. Dieses Protokoll beschreibt die Dissoziation von einem Erwachsenen testis; Volumes können dementsprechend für Jugendliche oder für zusätzliche Hoden angepasst werden. 1. Testis Dissoziation <ol><li> Legen Sie eine decapsulated Hoden in einen 15-ml-Tube. </li><li> Add 3-ml Gey-Balance-Salzlösung (GBSS) mit 120 U / ml Collagenase Typ I. </li><li> Add 10 ul DNAse I (1mg/ml Stammlösung in 50% Glycerin) und schütteln Sie sie kräftig von Hand, bis Sie Hoden Tubuli beginnen distanzieren sehen. </li><li> Agitieren horizontal auf einen Höchstbetrag von 120 rpm für 15 min bei 33 ° C. </li><li> Dekantieren für 1 min vertikal bei Raumtemperatur und Überstand verwerfen. </li><li> Wiederholen Sie die Schritte 1,2 bis 1,5. </li><li> Fügen Sie 2,5 ml GBSS mit Collagenase Typ I, 50 ul einer 50mg/ml Trypsin-Stammlösung in 1 mM HCl-Lösung, und 10 ul DNAse I (1mg/ml) resuspendiert und das Röhrchen mehrere Male. </li><li> Agitieren horizontal auf einen Höchstbetrag von 120 rpm für 15 min bei 33 ° C. </li><li> Mit Kunststoff-Einweg-Pasteur-Pipette mit breiter Öffnung, Pipette vorsichtig nach oben und unten für 3 min.No Klumpen sichtbar sein soll an dieser Stelle. </li><li> Add 30 ul Trypsin, 10 ul DNAse I, 40 ul Hoechst 33342 in DMSO (10 mg / ml) resuspendiert und das Röhrchen mehrere Male. </li><li> Agitieren horizontal auf einen Höchstbetrag von 120 rpm für 15 min bei 33 ° C. </li><li> Add 400-ul von fötalem Kälberserum (FCS) und mischen durch Invertieren, um Trypsin zu inaktivieren. </li><li> Finale Färbung wird durch Zugabe von 50 ul Hoechst 33342 resuspendiert in DMSO (10 mg / ml), und 10 ul DNAse I (1 mg / ml) durchgeführt. </li><li> Agitieren horizontal auf einen Höchstbetrag von 120 rpm für 15 min bei 33 ° C. </li><li> Die dissoziierten testis Probe wird dann durch zwei 40-um GBSS weitergegeben vorbefeuchtet Einwegfiltern über einen 50-ml konischen Rohr, dann 5 ul von Propidiumiodid (PI)-Lösung hinzugefügt und Probe wird vorsichtig gemischt mit einer Pipette mehrmals mit Einweg-Pasteur Pipette. </li><li> Die Probe wird auf einer 5-ml Kunststoff-Spritze durch eine 18-Gauge-Nadel übertragen. Letzteres wird durch eine 22-Gauge-Nadel für die Probenvorbereitung Lieferung ersetzt. Die Spritze wird auf Eis gelagert und geschützt vor Licht, bis sie zur FACS Verarbeitung. </li></ol> 2. FACS-Setup und Reinigung <ol><li> Sorting wurde auf einem Becton-Dickinson Aria IIU Zellsortierer durchgeführt. Die beschriebenen Bedingungen sollten daher als Ausgangspunkt verwendet werden, insbesondere wenn mit unterschiedlicher Ausrüstung. Wir haben oben beschriebenen Bedingungen gelten 7,8. </li><li> Da die Aria IIU keinen UV-Laser, passten wir das Protokoll zum Nachweis von Hoechst-Färbung mit einem 405nm violetten Laser zusätzlich verwendeten wir eine 488nm blauen Laser für Vorwärts-und Seitwärts-Streulicht-Detektion. Darüber hinaus wurden einige Filter modifiziert, um einige rote Laser Undichtigkeit was zu einer verbesserten Streudiagramm Schärfe zu begrenzen. </li><li> Die violetten Laser mit einer 450/40nm Bandpass für den Nachweis von Hoechst Blau-Emission und eine 585/42nm Bandpass für Hoescht Red Emission konfiguriert. A 502nm langen Pass wurde zur Trennung von Blau von roter Fluoreszenz. </li><li> A 100 pm Düse wurde mit einem Tropfen Antriebsfrequenz von 28.000 Tropfen / Sekunde. Die Probe Schwelle lag bei rund 4000 Veranstaltungen / Sekunde. Die Temperaturregelung Option wurde für die Probenahme und Sammlung Röhrchen bei 4 ° C zu halten während der gesamten Dauer der Sortierung. Darüber hinaus wurde die Probe Agitation Feature bei 200 Umdrehungen pro Minute verwendet werden, um die Probe aus sedimentierenden der ganzen Art zu verhindern. </li><li> In der Regel wurden zwei vor drei Hoden pro 6 Stunden sort verarbeitet ermöglicht die Sammlung von 0,5-2.0x10 6 Zellen für jede Population (siehe repräsentativen Abschnitt). </li><li> Die Probe wurde in Aliquots von ca. 750μl aus der Spritze verzichtet sortiert. In der Zwischenzeit, während dieser Pausen die Sammlung Röhrchen wurden bei 4 ° C, geschützt vor Licht und vorsichtig gemischt vor der Wiederaufnahme zu sortieren. </li><li> Die sortierten Proben wurden in einem 12x75mm Glas Borosilikatglas Sammlung Röhrchen mit 250μl DMEM mit 10% FCS gesammelt. </li></ol> 3. Repräsentative Ergebnisse: Ein typisches FACS-Profil ist in Abbildung 1 dargestellt. Alle wichtigen Schritte der Meiose sind identifiziert und werden in der Legende angegeben. Wenn der Hoechst 33342-Färbung nicht optimal ist, wird das globale Profil erscheinen wesentlich kompakter und weniger definiert. Ein häufiges Problem hinsichtlich nicht ausreichender Zugabe von DNAse, die eine schnelle Zelle Verklumpung auslösen wird. Die Zugabe von zusätzlichen DNAse überhaupt angeklagt Schritte in der Regel löst dieses Problem. Freeze-thawing der DNAse Lager (bei -20 ° C) sollte auf ein Minimum reduziert werden, da dies zu einer raschen Verlust der Enzymaktivität. Spo11 ist der meiotischen Endonuklease, Doppelstrang-Brüche an den Standorten der meiotischen Rekombination Hotspots 12 richtet. Abbildung 2 zeigt typische Profile eines Spo11-null Hoden, wo Doppelstrangbruch nicht ausgebildet sind, was zu einer gescheiterten Meiose 12, sowie eine pre-puberous Profil eines 13-Tage alt Maus, die asynchrone Natur von dieser ersten Welle der Meiose zeigt. Standard-zytogenetische Analysen unter Verwendung der Kombination von synaptonemal komplexe Protein 3 (SCP3) und phosphoryliert Histon-Antikörper H2AX stage-spezifischen Meiose Marker sollten zunächst verwendet werden, um bestätigt die Reinheit und die Art der Sortierung meiotischen Zellen, wie beschrieben Patentrechtsgelehrte 11. <img src="/files/ftp_upload/2602/2602fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Wildtyp-Meiose FACS-Profilen. A) Repräsentative Streudiagramm dargestellt. Die gated Zellen gezeigt werden. Beachten Sie die große Menge an Schutt in erwachsenen Hoden, die hauptsächlich aus leeren Membranen und Spermatide Schwänze. B) Ein Vertreter PI Plot zeigt die sehr begrenzte Menge an PI-positiven Zellen in einer Probe vorhanden. Die typische Gate wird gezeigt. C) Ein Vertreter Wildtyp-Hoechst 33342 FACS-Profil angezeigt wird. Die verschiedenen meiotischen Zellpopulationen, die unter Verwendung dieser Methode für weitere Untersuchungen werden können, sind angegeben: Spermatogonien (Sp), Pre-Leptotän (PL), Leptotän-Zygotän (L / Z), frühe-Pachytän (EP), Mitte-Pachytän ( MP), Late-Pachytän (LP), Diplotän (D), und runden Spermatiden (RS). <img src="/files/ftp_upload/2602/2602fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Spo11-null-und Wildtyp-Tag 13 meiotischen FACS-Profilen. A) Ein typischer Spo11-null-Profil ist mit einer klaren meiotischen Versagen, in denen keine Bühne hinter dem Leptotän / Zygotän frühen Pachytän erkannt werden können dargestellt. B) Das Profil eines 13-Tage alten Wildtyp-männliche Maus zeigt die hohe Konzentration von Leptotän / Zygotän Zellen. Diese erste Welle der Meiose ist ziemlich asynchron, wie übersichtlich visualisiert mit dieser Methode.

<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>

Das Protokoll hier vorgestellte erlaubt es, gleichzeitig von ausgewachsenen männlichen Mäusen reinigen das gesamte Spektrum der meiotischen Phase-Zellen mit sehr hoher Reinheit, so dass Forscher, die Dynamik dieser grundlegenden Prozess zu studieren. Gereinigte Zellen können für zahlreiche Anwendungen von RNA-Extraktion 9,10, nucleosome Mapping 11, rekombinantes Molekül-Erkennung, Protein-Analysen und vieles mehr verwendet werden. Allerdings haben Nachweismethoden auf die Menge an meiotischen ...

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<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>
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<td>6" Transfer Pipet</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>137119D</td>
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flow cytometry purification of mouse meiotic cells

Die Heterogenität der Zelltypen in den Hoden und das Fehlen von meiotischen Zellkultur-Modellen erhebliche Hürden für die Untersuchung der eindeutigen Unterscheidung Programme, die offenkundig sind während der Meiose worden. Zwei wesentliche Methoden wurden entwickelt, um zu reinigen, in unterschiedlichem Ausmaß, verschiedenen meiotischen Fraktionen von Erwachsenen und Jungtieren: Elutriation oder Staput (Sedimentation) mit BSA und / oder Percoll-Gradienten. Beide Methoden beruhen auf Zellgröße und Dichte zu trennen meiotischen Zellen 1-5. Insgesamt, abgesehen von wenigen Zellpopulationen 6, scheitern diese Protokolle, um eine ausreichende Reinheit der zahlreichen meiotischen Zellpopulationen, die für die detaillierte molekulare Analysen ergeben. Darüber hinaus mit solchen Methoden in der Regel eine Art von meiotischen Zellen können zu einer bestimmten Zeit, die eine zusätzliche Ebene der Komplexität in Bezug auf die Reproduzierbarkeit und Homogenität beim Vergleich meiotischen Zellproben fügt gereinigt werden. Hier beschreiben wir eine raffinierte Methode, die man einfach visualisieren, zu identifizieren und zu reinigen meiotischen Zellen erlaubt, die aus Keimzellen zu runden Spermatiden, mit FACS mit Hoechst 33342 Färbung 7,8 kombiniert. Diese Methode bietet eine umfassende Momentaufnahme des gesamten meiotischen Prozess und ermöglicht es, hoch zu reinigen lebensfähige Zellen aus den meisten Stadium der Meiose. Diese gereinigten Zellen können dann im Detail für die molekulare Veränderungen, die Progression durch Meiose zu begleiten, zum Beispiel Veränderungen in der Genexpression 9,10 und die Dynamik der Nukleosomen Belegung an Hotspots der meiotischen Rekombination 11 analysiert werden.

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

Flow Cytometry Purification of Mouse Meiotic Cells

Eine effiziente Methode, um hochreine lebensfähig meiotischen Fraktionen aus Maus Hoden zu erhalten, ist beschrieben, die vereint ein raffiniertes Zelle Dissoziation Protokoll mit fluoreszierenden activated cell sorting (FACS). Diese Methode nutzt die Unterschiede in der DNA-Gehalt und nukleare Dichte der diskreten meiotischen Fraktionen.

Durchflusszytometrie Reinigung der Maus meiotischen Zellen

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 Aufrufe.  The Scripps Research Institute. Eine effiziente Methode, um hochreine lebensfähig meiotischen Fraktionen aus Maus Hoden zu erhalten, ist beschrieben, die vereint ein raffiniertes Zelle Dissoziation Protokoll mit fluoreszierenden activated cell sorting (FACS). Diese Methode nutzt die Unterschiede in der DNA-Gehalt und nukleare Dichte der diskreten meiotischen Fraktionen.

Serie: 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.

Färbung von Proteinen in Gelen mit Coomassie G-250 ohne organische Lösungsmittel und Essigsäure

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

Forschung

Biologie

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.

Durchflusszytometrie-basierte Reinigung von<em&gt; S. cerevisiae</em&gt; Zygotes

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.

Optimiert Färbung und Proliferation Modeling-Methoden für die Zellteilung Überwachung mit 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 ...

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.

Cytosolischen Calcium Messungen in Nierenepithelzellen mittels Durchflusszytometrie

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.

Meiotischen Spindel Assessment in Maus-Oozyten von siRNA-vermittelte Silencing

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.

LED Thermo Flow - Die Kombination mit Optogenetik Flow Cytometry

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.

Vorbereitung der meiotischen Chromosom erstreckt sich von Maus Spermatozyten

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.

Analyse von Zellsuspensionen isoliert von festen Geweben von 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 ...

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-innere Zelle Masse Injektion von embryonalen Stammzellen führt zu höheren Chimerism Raten

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.

Messung des mitochondrialen Masse- und Membranpotenzials in hämatopoetischen Stammzellen und T-Zellen durch Durchflusszytometrie

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