Este projecto foi apoiado em parte por verbas do Estado da Flórida para Scripps e os números de prêmio R01GM085079 R21HD061304 e do Instituto Nacional de Ciências Médicas Gerais e do Instituto Nacional de Saúde Infantil e Desenvolvimento Humano, respectivamente. Este é o número 20917 manuscrito de The Scripps Research Institute.

Este protocolo pode ser separado em duas etapas principais: (1) a dissociação e Hoechst 33342 coloração de células de camundongo testículos seguido, se necessário, (2) FACS triagem das frações relevantes meiótica, incluindo todas as fases da meiose, a partir de células germinativas de espermátides arredondadas. Depois de coletados, essas populações altamente enriquecido meiótica pode ser usado para uma ampla gama de análise. Este protocolo descreve a dissociação de um testículo adulto; volumes pode ser adaptado para os jovens ou para testes adicionais. 1. Testículo dissociação <ol><li> Coloque um testículo decapsulated em um tubo de 15 ml. </li><li> Adicionar 3 ml de Salt Gey do Balance Solução (GBSS) contendo 120 U / ml de colagenase tipo I. </li><li> Adicionar 10 ml de DNAse I (1mg/ml solução estoque em glicerol 50%) e agite-a vigorosamente com a mão até que você veja testicular túbulos começando a dissociar. </li><li> Agitar horizontalmente em um máximo de 120 rpm por 15 min a 33 ° C. </li><li> Decantar por 1 min verticalmente em temperatura ambiente e descartar o sobrenadante. </li><li> Repita os passos de 1,2-1,5. </li><li> Adicionar 2,5 ml de colagenase GBSS contendo o tipo I, 50 ul de uma solução de 50mg/ml de ações Tripsina ressuspenso em solução de HCl 1mM, e 10 ml de DNAse I (1mg/ml), e inverter o tubo várias vezes. </li><li> Agitar horizontalmente em um máximo de 120 rpm por 15 min a 33 ° C. </li><li> Usando plástico descartáveis ​​pipeta Pasteur com orifício de largura, pipeta suavemente para cima e para baixo por três aglomerados min.No deve ser visível neste momento. </li><li> Adicionar 30 mL de tripsina, 10 ml de DNAse I, 40 mL de Hoechst 33342 ressuspenso em DMSO (10 mg / ml), e inverter o tubo várias vezes. </li><li> Agitar horizontalmente em um máximo de 120 rpm por 15 min a 33 ° C. </li><li> Adicionar 400 mL de soro fetal bovino (FCS) e misturar por inversão para inativar a tripsina. </li><li> Coloração final é realizada pela adição de 50 ul de Hoechst 33342 ressuspenso em DMSO (10 mg / ml) e 10 ml de DNAse I (1 mg / ml). </li><li> Agitar horizontalmente em um máximo de 120 rpm por 15 min a 33 ° C. </li><li> A amostra é então dissociado testículo passou por duas GBSS 40 mícrons de pré-filtros descartáveis ​​molhada ao longo de um tubo cônico de 50 ml, então mL 5 de iodeto de propídio (PI) é adicionado e solução amostra é misturada delicadamente por pipetagem várias vezes com Pasteur descartáveis pipeta. </li><li> Amostra é transferida para uma seringa de plástico de 5 ml por meio de uma agulha de calibre 18. O último é substituído por uma agulha de calibre 22 para a entrega da amostra. A seringa é armazenado em gelo e protegido da luz até que esteja pronto para o processamento FACS. </li></ol> 2. FACS Setup e Purificação <ol><li> Classificação foi realizada em um Becton-Dickinson classificador célula Aria IIU. As condições descritas deve ser utilizada como ponto de partida, especialmente quando se usa equipamentos diferentes. Usamos condições anteriormente descritas 7,8. </li><li> Desde o IIU Aria não tem um laser UV, adaptamos o protocolo para a detecção de coloração Hoescht usando um laser violeta 405nm além usamos um laser de 488nm azul para a detecção de dispersão para a frente e lateral. Além disso, alguns filtros foram modificadas, a fim de limitar algumas leakiness laser vermelho, resultando em nitidez enredo melhorou dispersos. </li><li> O laser violeta foi configurado com um passa-banda 450/40nm para a detecção de Hoescht Azul de emissão e um passa-banda 585/42nm para Hoescht emissão Vermelho. Um passe longo 502nm foi utilizado para separar o azul do fluorescência vermelha. </li><li> Um bico M 100 foi usada com uma freqüência disco gota de 28.000 gotas / segundo. A taxa de limiar de amostra foi de aproximadamente 4000 eventos / segundo. A opção de controle de temperatura foi usado para manter a amostra e tubos de coleta a 4 ° C todo o período de classificação. Além disso, o recurso de agitação da amostra foi utilizado em 200 rpm para evitar que a amostra de sedimentação em todo o tipo. </li><li> Normalmente, 02:58 testículos foram processados ​​por seis horas espécie permitindo a coleta de 0,5-2.0x10 6 células de cada população (ver secção representativa). </li><li> A amostra foi classificada em alíquotas de aproximadamente 750μl dispensado da seringa. Enquanto isso, durante estas pausas a tubos de coleta foram mantidas a 4 ° C, protegido da luz, e gentilmente misturado antes da espécie de reinício. </li><li> As amostras classificadas foram coletadas em um tubo de coleta de vidro 12x75mm borosilicato contendo 250μl DMEM suplementado com 10% SFB. </li></ol> 3. Resultados representativos: Um perfil FACS típico é mostrado na Figura 1. Todos os passos principais da meiose são identificados e são indicadas na legenda. Se a Hoechst 33342 mancha não é ideal, o perfil global vai aparecer muito mais compacto e menos definida. Um problema comum que diz respeito não a adição de DNAse suficiente, o que irá induzir aglutinação celular rápida. Adição de DNAse extras em todas as etapas indiciado geralmente resolve este problema. ª congelarawing do estoque de DNAse (armazenadas a -20 ° C) deve ser reduzido ao mínimo, pois isso resulta em rápida perda de atividade enzimática. Spo11 é a endonuclease meiótica que direciona dupla vertente quebra em locais de hotspots de recombinação meiótica 12. A Figura 2 mostra perfis típicos de um testículo Spo11 nulo onde fita dupla ruptura não são formadas, resultando em uma meiose abortiva 12, bem como um pré-puberous perfil de um rato de 13 dias de idade, o que revela a natureza assíncrona da primeira onda de meiose. Análises citogenéticas padrão usando a combinação de proteínas complexas sinaptonêmico 3 (SCP3) e fosforilado anticorpos H2AX histona estágio marcadores específicos de meiose devem ser inicialmente utilizada para confirmar a pureza ea natureza das células classificadas meiótica, como descrito elswhere 11. <img src="/files/ftp_upload/2602/2602fig1.jpg" alt="Figura 1" /> Figura 1. FACS tipo selvagem perfis meiose. A) gráfico de dispersão Representante é mostrado. As células gated são mostrados. Observe a grande quantidade de detritos presentes no testículo adulto contendo principalmente membranas vazio e caudas espermátides. B) A trama mostra o PI representante quantidade muito limitada de células PI positivos presentes em uma amostra. O portão típico é mostrado. C) Um representante do tipo selvagem Hoechst 33342 perfil FACS é mostrado. As várias populações de células meióticas que pode ser purificado usando este método para um estudo mais aprofundado são indicadas: espermatogônias (Sp), pré-leptóteno (PL), leptóteno-Zigóteno (L / Z), early-Paquíteno (PE), de meia-Paquíteno ( MP), late-Paquíteno (LP), diplóteno (D), e volta espermátides (RS). <img src="/files/ftp_upload/2602/2602fig2.jpg" alt="Figura 2" /> Figura 2. Spo11 nulo e do tipo selvagem dia 13 perfis meiótica FACS. A) Um perfil Spo11 nulo típico é mostrado com uma clara falha meiótica, onde nenhum momento após o leptóteno / Zigóteno início de paquíteno pode ser detectado. B) O perfil de um de 13 dias de idade do rato do tipo selvagem macho mostra a alta concentração de células leptóteno / Zigóteno. Esta primeira onda da meiose é bastante assíncronas, como claramente visualizado utilizando esta 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>

Não há conflitos de interesse declarados.

O protocolo aqui apresentado permite, simultaneamente, purificar a partir de camundongos machos adultos toda a gama de células estágio meiótico com pureza muito elevado, permitindo que os pesquisadores para estudar a dinâmica deste processo fundamental. Células purificadas podem ser usados ​​para inúmeras aplicações que vão desde extração de RNA 9,10, mapeamento nucleosome 11, detecção molécula recombinante, análises de proteínas, e muitos mais. No entanto, métodos de detecção devem ser adaptadas à quantidade de células meióticas purificada. Além disso, com a alta reprodutibilidade deste método, é possível agrupar vários tipos independente de a mesma fração realizadas em dias diferentes para aumentar a quantidade de material necessário para uma experiência particular. Além disso, este método fornece investigadores com uma representação visual única de todo o processo meiótico, o que permite avaliar rapidamente os efeitos do nocaute genético, a aberração progressão do desenvolvimento, ou o efeito de qualquer tratamento especial destinado a atentar contra a meiose (por exemplo, a irradiação, ou tratamento com inibidores de molécula pequena). Passos críticos neste protocolo são: (i) o uso de DNAse suficiente, o que facilita muito a separação de células, evitando aglomerações de células e agregados, e (ii) consistente coloração Hoechst 33342, que é necessário distinguir eficientemente as várias populações de células estágio meiótico. Além disso, a operadora FACS devem se familiarizar com as especificidades e peculiaridades de separação de células meióticas, a fim de melhor forma seu instrumento de instalação para obter os perfis mostrados na Figura 1 e detalhadas no vídeo. Uma modificação possível seria substituir a Hoechst 33342 com o novo Violet DyeCycle Vybrant Stain (Invitrogen), que foi otimizado para o laser violeta. No entanto, o perfil geral não seria de esperar para mudar drasticamente. Finalmente, é também importante notar que devido à natureza do tecido usado, um procedimento típico terá 1 ½ hr de preparar as células meióticas e de 4 a 6 horas para o tipo de células, além de adicional de pós-tipo de manipulações.

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		<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>
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flow cytometry purification of mouse meiotic cells

A natureza heterogênea de tipos de células nos testículos e na ausência de modelos de cultura de células meióticas foram obstáculos significativos para o estudo dos programas de diferenciação únicos que se manifestam durante a meiose. Dois principais métodos foram desenvolvidos para purificar, em diferentes graus, várias frações meiótica de ambos os adultos e animais jovens: elutriação ou Staput (sedimentação), utilizando BSA e / ou gradientes de Percoll. Ambos os métodos dependem do tamanho das células e densidade para separar células meióticas 1-5. No geral, exceto para populações de células poucos 6, estes protocolos não ceder pureza suficiente das inúmeras populações de células meióticas que são necessários para análises detalhadas molecular. Além disso, com tais métodos geralmente um tipo de células meióticas podem ser purificada em um determinado momento, o que adiciona um nível extra de complexidade quanto à reprodutibilidade e homogeneidade quando se comparam amostras de células meióticas. Aqui, nós descrevemos um método refinado que permite facilmente visualizar, identificar e purificar as células meióticas, a partir de células germinativas de espermátides arredondadas, usando FACS combinado com coloração Hoechst 33342 7,8. Este método oferece um instantâneo global de todo o processo meiótico e permite altamente purificar as células viáveis ​​da maioria fase da meiose. Estas células purificadas podem ser analisados ​​em detalhe para mudanças moleculares que acompanham a progressão através da meiose, por exemplo, mudanças na expressão gênica 9,10 ea dinâmica de ocupação nucleosome em hotspots de recombinação meiótica 11.

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

Flow Cytometry Purification of Mouse Meiotic Cells

Um método eficiente para obter altamente purificada viável frações meiótica de testículo do rato é descrito, que combina uma refinada célula dissociação protocolo com fluorescentes separação de células ativadas (FACS). Este método tira vantagem de diferenças no conteúdo de DNA nuclear e densidade de frações discretas meiótica.

Purificação de Citometria de Fluxo de células de camundongo meiótica

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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Meiosis

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17.6K visualizações.  The Scripps Research Institute. Um método eficiente para obter altamente purificada viável frações meiótica de testículo do rato é descrito, que combina uma refinada célula dissociação protocolo com fluorescentes separação de células ativadas (FACS). Este método tira vantagem de diferenças no conteúdo de DNA nuclear e densidade de frações discretas meiótica.

Vídeo: 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.

Coloração de proteínas em géis com Coomassie G-250 sem ácido acético e Solventes Orgânicos

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

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.

Purificação Citometria de Fluxo com base em<em&gt; S. cerevisiae</em&gt; Zigotos

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.

Optimizado coloração e Proliferação Métodos de Modelagem para Monitoramento Divisão Celular usando Corantes Rastreamento celulares

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.

Medidas cálcio citosólico em células epiteliais renais por citometria de fluxo

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 Avaliação de fuso em oócitos mouse por silenciamento mediado por siRNA

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 - Combinando optogenética com Citometria de Fluxo

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.

Preparação de cromossomos se espalha a partir do Mouse espermatócitos

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.

Análise de suspensões celulares isoladas de tecidos sólidos por citometria de fluxo espectral

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-interno pilha injeção maciça de células-tronco embrionárias leva a taxas mais elevadas de quimerismo

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.

Medição da massa mitocondrial e potencial de membrana em células-tronco hematopoiéticas e células T por citometria flow

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