Ce projet a été soutenu en partie par l&#39;argent de l&#39;Etat de Floride aux Scripps et les numéros de récompense et de R01GM085079 R21HD061304 de l&#39;Institut national des sciences médicales générales et l&#39;Institut national de la santé infantile et du développement humain, respectivement. Ceci est le numéro de manuscrit de 20917 The Scripps Research Institute.

Ce protocole peut être séparé en deux grandes étapes: (1) la dissociation et Hoechst 33342 coloration des cellules des testicules de souris suivie, si nécessaire, (2) FACS de tri des fractions pertinentes méiotique, y compris toutes les étapes de la méiose, à partir de cellules germinales au spermatides rondes. Une fois collectées, ces populations hautement enrichi méiotique peut être utilisé pour un large éventail d&#39;analyses. Ce protocole décrit la dissociation d&#39;un testicule adulte; volumes peuvent être adaptées en conséquence pour les mineurs ou pour les testicules supplémentaires. 1. Testicule dissociation <ol><li> Placez un testicule décapsulé dans un tube de 15 ml. </li><li> Ajouter 3 ml de solution de Gey équilibre du sel (GBSS) contenant 120 U / ml de collagénase de type I. </li><li> Ajouter 10 ul de DNAse I (1mg/ml solution stock de glycérol 50%) et agiter vigoureusement à la main jusqu&#39;à ce que vous voyez tubules testiculaires commencent à se dissocier. </li><li> Agiter horizontalement à un maximum de 120 rpm pendant 15 min à 33 ° C. </li><li> Décanter pendant 1 min à la verticale à la température ambiante et éliminer le surnageant. </li><li> Répétez les étapes 1.2 à 1.5. </li><li> Ajouter 2,5 ml de GBSS contenant la collagénase de type I, 50 ul d&#39;une solution de trypsine stocks 50mg/ml remises en suspension dans une solution d&#39;HCl 1mM, et 10 pi de DNAse I (1mg/ml), et inverser le tube plusieurs fois. </li><li> Agiter horizontalement à un maximum de 120 rpm pendant 15 min à 33 ° C. </li><li> L&#39;utilisation de plastique jetables pipette Pasteur avec large orifice, une pipette doucement de haut en bas pour les touffes min.No 3 devrait être visible à ce stade. </li><li> Ajouter 30 ul de la trypsine, 10 pl de la DNAse I, 40 pl de Hoechst 33342 resuspendues dans du DMSO (10 mg / ml), et inverser le tube plusieurs fois. </li><li> Agiter horizontalement à un maximum de 120 rpm pendant 15 min à 33 ° C. </li><li> Ajouter 400 ul de sérum de veau foetal (FCS) et mélanger en retournant pour inactiver la trypsine. </li><li> Coloration finale est effectuée en ajoutant 50 pl de Hoechst 33342 resuspendues dans du DMSO (10 mg / ml), et 10 pi de DNAse I (1 mg / ml). </li><li> Agiter horizontalement à un maximum de 120 rpm pendant 15 min à 33 ° C. </li><li> L&#39;échantillon est ensuite dissocié testicule passé par deux GBSS 40 um préhumidifié filtres jetables sur un tube conique de 50 ml, puis 5 ul d&#39;iodure de propidium (PI) et la solution est ajoutée de l&#39;échantillon est mélangé doucement par pipetage plusieurs fois avec Pasteur jetables pipette. </li><li> L&#39;échantillon est transféré dans une seringue en plastique de 5 ml à travers une aiguille de calibre 18. Cette dernière est remplacée par une aiguille de calibre 22 pour la livraison d&#39;échantillons. La seringue est stocké sur la glace et protégé de la lumière jusqu&#39;au moment de son traitement de FACS. </li></ol> 2. Configuration FACS et purification <ol><li> Le tri a été effectué sur un trieur Becton-Dickinson Aria cellules IIU. Les conditions décrites doivent donc être utilisés comme point de départ en particulier lors de l&#39;utilisation des équipements différents. Nous avons utilisé les conditions décrites précédemment 7,8. </li><li> Depuis l&#39;IIU Aria n&#39;a pas un laser UV, nous avons adapté le protocole pour la détection de coloration Hoescht en utilisant un laser violet 405nm en plus nous avons utilisé un laser 488nm bleu pour la détection diffusion vers l&#39;avant et latérale. Par ailleurs, certains filtres ont été modifiées afin de limiter certains perméabilité laser rouge résultant de la netteté améliorée complot dispersés. </li><li> Le laser violet a été configuré avec une bande passante 450/40nm pour la détection de Hoescht Bleu d&#39;émission et un passe-bande pour les 585/42nm Hoescht-Rouge émission. Un laissez-passer 502nm long a été utilisé pour séparer bleue de fluorescence rouge. </li><li> Une buse 100 um a été utilisé avec une fréquence d&#39;entraînement goutte de 28000 gouttes / seconde. Le taux seuil de l&#39;échantillon était d&#39;environ 4000 événements / seconde. L&#39;option de contrôle de température a été utilisée pour maintenir l&#39;échantillon et les tubes de prélèvement à 4 ° C toute la durée de tri. En outre, la fonctionnalité de l&#39;agitation de l&#39;échantillon a été utilisé à 200 rpm pour empêcher l&#39;échantillon de sédimentation dans le tri. </li><li> Habituellement, deux à trois testicules ont été traités par 6 heures de tri permettant la collecte de 0,5-2.0x10 6 cellules pour chaque population (voir la section représentant). </li><li> L&#39;échantillon a été trié dans des aliquotes d&#39;environ 750μl dispensé de la seringue. En attendant, pendant ces pauses les tubes de prélèvement ont été conservés à 4 ° C, protégé de la lumière, et mélanger doucement avant de reprendre de tri. </li><li> Les échantillons ont été collectés triés dans un tube de collecte du verre borosilicaté contenant 250μl 12x75mm DMEM supplémenté avec 10% de FCS. </li></ol> 3. Les résultats représentatifs: Un profil typique FACS est montré dans la figure 1. Toutes les grandes étapes de la méiose sont identifiés et sont indiqués dans la légende. Si le Hoechst 33342 tache n&#39;est pas optimale, le profil global apparaîtra beaucoup plus compact et moins bien définies. Un problème commun concerne non ajoutant suffisamment DNAse, qui va induire l&#39;agglutination cellulaire rapide. Ajout d&#39;extra DNAse à toutes les étapes inculpé résout généralement ce problème. E gelAwing du stock DNAse (conservés à -20 ° C) devrait être maintenu à un minimum, car cela entraîne une perte rapide de l&#39;activité enzymatique. Spo11 est l&#39;endonucléase méiotique qui dirige des cassures double brin dans des sites de hotspots recombinaison méiotique 12. La figure 2 montre les profils typiques d&#39;un testicule Spo11-nulle, où cassures double brin ne sont pas formés, résultant en une méiose avortée 12, ainsi que d&#39;un pré-puberous profil d&#39;une souris de 13 jours, ce qui révèle la nature asynchrone de cette première vague de la méiose. Analyses cytogénétiques standard en utilisant la combinaison de protéines complexes synaptonémal 3 (SCP3) et phosphorylée anticorps histone H2AX stade de la méiose des marqueurs spécifiques doivent être d&#39;abord utilisé pour confirmer la pureté et la nature des cellules triées méiotique, comme décrit elswhere 11. <img src="/files/ftp_upload/2602/2602fig1.jpg" alt="Figure 1" /> Figure 1. Wild-type de la méiose FACS profils. A) nuage de points représentant est montré. Les cellules fermée sont représentés. Notez la grande quantité de débris présents dans le testicule adulte contenant principalement des membranes vides et les queues des spermatides. B) Un tracé représentatif PI indique la quantité très limitée de cellules PI positives présentes dans un échantillon. La porte typique est montré. C) Un représentant de type sauvage Hoechst 33342 profil FACS est montré. Les différentes populations de cellules méiotiques qui peuvent être purifiés en utilisant cette méthode pour une étude plus approfondie sont indiqués: les spermatogonies (Sp), pré-leptotène (PL), leptotène-zygotène (L / Z), début Pachytène (EP), le Moyen-Pachytène ( spermatides MP), fin-Pachytène (LP), diplotène (D) et ronde (RS). <img src="/files/ftp_upload/2602/2602fig2.jpg" alt="Figure 2" /> Figure 2. Spo11-nulle et de type sauvage jours 13 méiotique profils FACS. A) Un typiques Spo11-nulle profil est représenté avec un échec patent méiotique, où aucune étape de la dernière leptotène / zygotène précoce pachytène peut être détecté. B) le profil d&#39;un 13-jours-anciens de type sauvage souris mâle montre la forte concentration de cellules leptotène / zygotène. Cette première vague de la méiose est plutôt asynchrone, aussi clairement visualisés en utilisant cette méthodologie.

<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>

Le protocole présenté ici permet un à la fois purifier de souris mâles adultes toute la gamme des cellules stade de la méiose avec une très grande pureté, ce qui permet aux enquêteurs d&#39;étudier la dynamique de ce processus fondamental. Cellules purifiées peuvent être utilisées pour de nombreuses applications allant de l&#39;extraction de l&#39;ARN 9,10, la cartographie nucléosome 11, la détection de molécules recombinantes, les analyses de protéines, et beaucoup plus. Cependant,...

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<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>
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flow cytometry purification of mouse meiotic cells

Le caractère hétérogène de types de cellules dans les testicules et l&#39;absence de modèles de culture cellulaire méiotique ont été des obstacles importants à l&#39;étude de la différenciation des programmes uniques qui se manifestent lors de la méiose. Deux principales méthodes ont été développées pour purifier, à des degrés divers, diverses fractions méiotique à la fois adulte et immature animaux: élutriation ou Staput (sédimentation) à l&#39;aide de BSA et / ou des gradients de Percoll. Ces deux méthodes reposent sur ​​la taille des cellules et la densité de séparer les cellules méiotiques 1-5. Dans l&#39;ensemble, sauf pour quelques populations de cellules 6, ces protocoles ne parviennent pas à donner une pureté suffisante des populations de nombreuses cellules méiotiques qui sont nécessaires pour des analyses détaillées moléculaire. Par ailleurs, avec de telles méthodes habituellement un type de cellules méiotiques peut être purifié à un moment donné, ce qui ajoute un niveau supplémentaire de complexité quant à la reproductibilité et l&#39;homogénéité en comparant des échantillons de cellules méiotiques. Ici, nous décrivons une méthode raffinée qui permet de visualiser facilement, d&#39;identifier et de purifier les cellules méiotiques, à partir des cellules germinales afin de spermatides rondes, en utilisant FACS combiné avec Hoechst 33342 coloration 7,8. Cette méthode fournit un instantané global de l&#39;ensemble du processus méiotique et permet de purifier les cellules viables hautement de la plupart des étapes de la méiose. Ces cellules purifiées peuvent alors être analysés en détail pour des changements moléculaires qui accompagnent la progression à travers la méiose, par exemple des changements dans les 9,10 expression des gènes et la dynamique de l&#39;occupation des nucléosomes sur les hotspots de la recombinaison méiotique 11.

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

Flow Cytometry Purification of Mouse Meiotic Cells

Une méthode efficace pour obtenir des fractions hautement purifiées viables méiotiques de la souris testicule est décrit, qui combine un cadre raffiné dissociation cellulaire protocole avec le tri de cellules fluorescentes (FACS). Cette méthode tire parti des différences dans le contenu en ADN et de la densité nucléaire de fractions distinctes méiotique.

Purification cytométrie en flux des cellules de souris méiotique

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

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17.5K vues.  The Scripps Research Institute. Une méthode efficace pour obtenir des fractions hautement purifiées viables méiotiques de la souris testicule est décrit, qui combine un cadre raffiné dissociation cellulaire protocole avec le tri de cellules fluorescentes (FACS). Cette méthode tire parti des différences dans le contenu en ADN et de la densité nucléaire de fractions distinctes méiotique.

Vidéo: 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.

Coloration des protéines dans les gels de Coomassie G-250, sans solvant et l&#39;acide acétique organique

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

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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.

Cytométrie en flux à base de purification de<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.

Optimisé méthodes de modélisation de coloration et de prolifération pour la surveillance division cellulaire en utilisant des colorants Suivi cellulaires

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.

Mesures de calcium cytosolique dans les cellules épithéliales rénales par cytométrie en flux

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

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.

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

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 - Optogenetics Combinant avec cytométrie de flux

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.

Préparation des chromosomes à la méiose se propage de Spermatocytes de souris

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 des suspensions de cellules isolées de tissus solides par Spectral cytométrie en flux

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-intérieur cellulaire massive Injection de cellules souches embryonnaires conduit à des taux plus élevés de chimérisme

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

Isolation and Characterization of the Immune Cells from Micro-dissected Mouse Choroid Plexuses

The brain is no longer considered as an organ functioning in isolation; accumulating evidence suggests that changes in the peripheral immune system can indirectly shape brain function. At the interface between the brain and the systemic circulation, the choroid plexuses (CP), which constitute the blood-cerebrospinal fluid barrier, have been highlighted as a key site of periphery-to-brain communication. CP produce the cerebrospinal fluid, neurotrophic factors, and signaling molecules that can shape brain homeostasis. CP are also an active immunological niche. In contrast to the brain parenchyma, which is populated mainly by microglia under physiological conditions, the heterogeneity of CP immune cells recapitulates the diversity found in other peripheral organs. The CP immune cell diversity and activity change with aging, stress, and disease and modulate the activity of the CP epithelium, thereby indirectly shaping brain function. The goal of this protocol is to isolate murine CP and identify about 90% of the main immune subsets that populate them. This method is a tool to characterize CP immune cells and understand their function in orchestrating periphery-to-brain communication. The proposed protocol may help decipher how CP immune cells indirectly modulate brain function in health and across various disease conditions.

This study uses flow cytometry and two different gating strategies on isolated perfused mice brain choroid plexuses; this protocol identifies the main immune cell subsets that populate this brain structure.

Isolement et caractérisation des cellules immunitaires des plexus choroïdes de souris micro-disséqués

The brain is no longer considered as an organ functioning in isolation; accumulating evidence suggests that changes in the peripheral immune system can ...

Isolation and Identification of Vascular Endothelial Cells from Distinct Adipose Depots for Downstream Applications

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone regulation, barrier functions, and angiogenesis. Endothelial cell dysfunction is a hallmark predictor and major driver for the progression of severe cardiovascular diseases, yet the underlying mechanisms remain poorly understood. The ability to isolate and perform analyses on endothelial cells from various vascular beds in their native form will give insight into the processes of cardiovascular disease. This protocol presents the procedure for the dissection of mouse subcutaneous and mesenteric adipose tissues, followed by isolation of their respective arterial vasculature. The isolated arteries are then digested using a specific cocktail of digestive enzymes focused on liberating functionally viable endothelial cells. The digested tissue is assessed by flow cytometry analysis using CD31+/CD45&#8722; cells as markers for positive endothelial cell identification. Cells can be sorted for immediate downstream functional assays or used to generate primary cell lines. The technique of isolating and digesting arteries from different vascular beds will provide options for researchers to evaluate freshly isolated vascular cells&#160;from arteries of interest and allow them to perform a wide range of functional tests on specific cell types.

This protocol details a method for the dissection of mouse adipose depots and the isolation and digestion of respective arteries to liberate and then identify the endothelial cell population. Freshly isolated cells used in downstream applications will advance the understanding of vascular cell biology and the mechanisms of vascular dysfunction.

Isolement et identification de cellules endothéliales vasculaires à partir de dépôts adipeux distincts pour des applications en aval

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone ...