Este trabalho foi financiado pelo Prêmio Inovação Eagles (Ordem Fraternal de Eagles) e pela Fundação Mayo.

** Este protocolo de vídeo é baseado em uma publicação associada 1: alta sensibilidade de detecção e análise quantitativa dos nativos interações proteína-proteína e complexos multiprotein por citometria de fluxo. Adam G. Schrum, Diana Gil, Elaine P. Dopfer, David L. Wiest, Laurence A. Turka, Wolfgang WA Schamel, Ed Palmer. STKE da ciência 2007 (389): PL2, 05 de junho de 2007, [DOI: 10.1126/stke.3892007pl2]. <a href="http://stke.sciencemag.org/cgi/content/abstract/2007/389/pl2">Clique aqui para ver esta publicação</a> . Antes de iniciar, preparar o seguinte stock Soluções aquosas: <table><tbody><tr><td> MES tampão de acoplamento: </td><td> Armazenar a 25 ° C </td></tr><tr><td> MES, pH 6,0 </td><td> 50 mM </td></tr><tr><td> EDTA </td><td> 1 mM </td></tr><tr><td> Têmpera / Bloqueio / Storage (SBQ) Buffer: </td><td> Armazenar a 4 ° C </td></tr><tr><td> BSA </td><td> 1% </td></tr><tr><td> Azida de sódio </td><td> 0,02% </td></tr><tr><td> PBS, pH 7,4 </td><td> 1x </td></tr><tr><td> Tampão FCM: </td><td> Armazenar a 4 ° C </td></tr><tr><td> Tris, pH 7,4 </td><td> 50 mM </td></tr><tr><td> NaCl </td><td> 100 mM </td></tr><tr><td> Azida de sódio </td><td> 0,02% </td></tr><tr><td> FBS </td><td> 5% </td></tr><tr><td> PBS, pH 7,4 </td><td> 1x </td></tr></tbody></table> Prepare imediatamente antes da utilização: <table><tbody><tr><td> EDAC-MES: </td><td> Dissolver 50 mg / mL em pó EDAC MES tampão de acoplamento </td></tr><tr><td> Digitonina solução (2% w / v): </td><td> Dissolva o pó digitonina em dH 2 O por aquecimento a 95 ° C por 5min, em seguida de resfriamento em gelo </td></tr><tr><td> Tampão de Lise: </td><td></td></tr><tr><td> Tris, pH 7,4 </td><td> 50 mM </td></tr><tr><td> NaCl </td><td> 150 mm </td></tr><tr><td> Solução digitonina </td><td> 1% </td></tr><tr><td> Inibidores da Protease </td><td> 1x </td></tr><tr><td> Manter no gelo </td><td></td></tr></tbody></table> 1. Acoplamento de mAb de Contas <ol><li> Determinar a concentração de contas do estoque adquirido por diluindo em PBS e contando com um hemocitômetro. Comece com uma diluição de 1:10000 de contas para a contagem. </li><li> Pipetar 18 x 10 6 contas em um tubo de microcentrífuga de 1,5 mL. </li><li> Lavar as contas 2 a 3 vezes em 0,5-1,0 mL de buffer Coupling MES, centrifugação a 20.000 g por 3 minutos a 25 ° C após cada lavagem. </li><li> Volte a suspender as contas em 50 mL de buffer Coupling MES. </li><li> Ativar os grupos carboxila nas contas de adição de 20 mL de recém-preparados EDAC-MES. </li><li> Misture delicadamente por 15 min a 25 ° C por manualmente pipetando para cima e para baixo. </li><li> Lave as contas ativadas 2 a 3 vezes em 0,5 a 1,0 mL PBS, centrifugação a 20.000 g por 3 minutos a 25 ° C após cada lavagem. </li><li> Volte a suspender as contas ativadas em 50 mL de PBS. </li><li> Adicionar 50 mL do mAb IP (a concentração de ações 0,2-1,0 mg / mL) à solução ativada talão. </li><li> Misture por 3 a 4 horas a 25 ° C, colocando o tubo em um shaker vibrando. Agitar o suficiente para impedir que a resolução das esferas no fundo do tubo. </li><li> Lavar mAb-coupled contas 2 a 3 vezes em 0,5 a 1,0 mL PBS, centrifugação a 20.000 g por 3 minutos a 25 ° C após cada lavagem. </li><li> Volte a suspender as contas em 100 mL de buffer SBQ. Estes podem ser armazenados a 4 ° C. </li><li> Suspendendo as contas assim, diluir 1:200-1:10,000 em PBS e contar com um hemocitômetro. A concentração deve ser medido para cada lote preparativa, de modo que o número de contas utilizadas em cada experimento IP ou pull-down pode ser controlada com precisão. </li></ol> 2. Pós-nuclear Preparação Lysate e Ip O método de lise e as condições de lise vai depender do tipo de célula e interações proteína-proteína em análise. Uma série de protocolos existem e podem ser modificados para sua aplicação. <ol><li> Lyse 20 x 10 6 &#39;pequena&#39; células, como linfócitos, ou 5 x 10 6 &#39;grande&#39; células, como macrófagos ou células tumorais, em 100 mL de buffer Lysis fresco em um tubo de microcentrífuga de 1,5 mL por 20min no gelo. Escala o volume de lise, conforme necessário. </li><li> Para remover núcleos e restos celulares insolúveis, centrifugar o lisado em 20.000 g por 10 min a 4 ° C. Manter o sobrenadante e descartar o pellet. </li><li> Adicionar 0,5 x 10 5 a 2,5 x 10 5 mAb-coupled contas ao lisado clarificado, pipetagem cuidadosamente para misturar. O volume mínimo que usamos pararealizar um único IP está a 5 mL. </li><li> Coloque em uma roda giratória verticais 4 horas durante a noite em uma sala fria. Definir a velocidade suficiente para impedir as contas de liquidação. É aceitável que o líquido de baixo volume de IPs para permanecer no fundo do tubo durante a rotação. </li></ol> 3. Sondagem da Bead-capturado Protein com fluorocromo-Anticorpos <ol><li> Lave as contas IP duas vezes em 0,2-1,0 mL gelada buffer FCM, centrifugação a 20.000 g a 4 ° C por 3 min após cada lavagem. </li><li> Volte a suspender as contas em 100 FCM Buffer e alíquota 20 mL mL em cada um dos cinco ou mais tubos de 1,5 mL de microcentrífuga ou poços de uma placa de fundo da chaminé. </li><li> Adicionar fluorocromo-mAbs às amostras. Executar a FCM mancha de acordo com instruções do fornecedor ou de acordo com as concentrações de determinados empiricamente. Como ponto de partida, adicione 0,2-1 mL de solução anticorpo estoque (em 0,2-1,0 mg / mL) por tubo ou bem, e incubar por 40 min no gelo. </li><li> Lavar sondado contas em 1,5 ml tubos duas vezes em 1,0 mL de buffer FCM gelada, centrifugação a 20.000 g a 4 ° C por 3 min após cada lavagem. Remova cuidadosamente o sobrenadante após cada lavagem, tendo o cuidado para não perturbar as contas. Por um prato de chaminé inferior, lave duas vezes com 0,2 mL gelada buffer FCM, centrifugação a 1000g por 5 min a 4 ° C após cada lavagem. Um pipetador multicanal é útil para esta etapa. Para remover o sobrenadante de contas em um prato fundo da chaminé, &#39;filme&#39; da placa de uma vez, então, ainda de cabeça para baixo, blot qualquer pinga das bordas dos poços. O volume residual em cada poço, contendo as contas, serão cerca de 20 mL. </li><li> Volte a suspender as contas em 200 mL de buffer FCM por amostra. Transferência para tubos rotulados FACS. Amostras estão agora prontos para FCM. </li></ol> 4. FCM Aquisição As contas CML descritos neste protocolo são 3-5 m de diâmetro, aproximadamente metade do diâmetro de um linfócito do mouse quiescente. Portanto, pode ser necessário aumentar manualmente o Forward Scatter (FSC) ganho ampères e Side Scatter (SSC) de tensão para que a população de eventos talão de registrar em escala. Contas individuais IP devem formar uma única população bem agrupado. As configurações e portão deve ser ajustado para excluir doublets talão e detritos. <ol><li> Antes de contas correntes ou padrões de talão, remova o adaptador de contenção de gotículas que circunda a entrada da amostra em alguns citómetros. Permitir que o fluido bainha a escorrer entre as amostras, pois isso irá limpar a entrada e evitar contas de serem transitadas de uma amostra para outra. </li><li> Use contas sem rótulo, um controle negativo de fluorescência, e Partículas Calibração Rainbow (PCRs), um controle positivo de fluorescência, para ajustar as configurações de modo que ambos os extremos negativos e positivos de fluorescência aparecem simultaneamente no log escala de eixo-x. Isso garante que a fluorescência das amostras experimentais também será on-escala. Note-se que PCRs são menores do que as contas da CML, para FSC e SSC parâmetros, bem como a posição portão, pode precisar ser temporariamente alteradas para que a amostra. </li><li> Return to-grânulo unlabeled configurações vez de calibração com PCRs é completo. Se apenas uma sonda fluorescente por amostra é usada, nenhuma compensação de fluorescência é necessário. Em geral, complexos multiprotein sonda com um único fluorocromo-mAb por amostra FCM, e examinamos vários parceiros de interação através da coloração paralelo amostras talão com mAbs específicos para as subunidades diferentes. </li><li> Adquirir e salvar arquivos de fluorescência de dados. Análise pode ser realizada utilizando software citometria de fluxo, tais como CellQuest, programa FlowJo, ou cflow. </li></ol> 5. Resultados representante <img src="/files/ftp_upload/2066/2066fig1.jpg" alt="Figura 1" /> Figura 1. Células IP FCM para TCR/CD3 complexo multiprotein. T a partir da estirpe camundongo BALB / c foram lisadas em digitonina 1%, eo lisado foi submetido a IP-FCM usando anti-CD3ε contas. Os complexos capturado contidas quantidades significativas de TCR-β (região roxo) e co-associados CD3-ε (verde), mas perto dos níveis de fundo (definida pela sonda imunoglobulina irrelevante, rosa trace) de outras proteínas, como Thy1.2, CD45, ou H-2K (d) (marrom, laranja e vestígios azul, respectivamente). Veja resultados semelhantes publicados anteriormente 1-6.

<ol>
	<li>Schrum, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-sensitivity+detection+and+quantitative+analysis+of+native+protein-protein+interactions+and+multiprotein+complexes+by+flow+cytometry.">High-sensitivity detection and quantitative analysis of native protein-protein interactions and multiprotein complexes by flow cytometry.</a> Sci STKE. , pl2-pl2 (2007).</li><li>Schrum, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+multiprotein+complexes+by+flow+cytometry.">Visualization of multiprotein complexes by flow cytometry.</a> Curr Protoc Immunol. Chapter 5, 9-9 (2009).</li><li>Teixeiro, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+division+and+death+are+segregated+by+mutation+of+TCRbeta+chain+constant+domains.">T cell division and death are segregated by mutation of TCRbeta chain constant domains.</a> Immunity. 21, 515-526 (2004).</li><li>Teixeiro, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+T+Cell+receptor+signals+determine+CD8++memory+versus+effector+development.">Different T Cell receptor signals determine CD8+ memory versus effector development.</a> Science. 323, 502-505 (2009).</li><li>Treves, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Junctate+is+a+key+element+in+calcium+entry+induced+by+activation+of+InsP3+receptors+and/or+calcium+store+depletion.">Junctate is a key element in calcium entry induced by activation of InsP3 receptors and/or calcium store depletion.</a> J Cell Biol. 166, 537-548 (2004).</li><li>Gil, D., Schrum, A. G., Alarcon, B., Palmer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+receptor+engagement+by+peptide-MHC+ligands+induces+a+conformational+change+in+the+CD3+complex+of+thymocytes.">T cell receptor engagement by peptide-MHC ligands induces a conformational change in the CD3 complex of thymocytes.</a> J Exp Med. 201, 517-522 (2005).</li></ol>

Informações sobre interações proteína-proteína é altamente relevante para a análise de muitos processos celulares, tais como transdução de sinal, a maturação linhagem, progressão do ciclo celular, apoptose e cascatas. FCM-IP oferece uma maneira rápida, quantitativa e sensíveis para analisar a interação de proteínas e definir os membros de complexos multiprotein em sua conformação nativa. Esferas podem ser acoplados e incubadas com lisados ​​celulares em um dia e pode ser sondado e analisadas no dia seguinte. Um formato de placa de 96 poços permite um grande número de amostras a serem analisadas de uma só vez para a coleta de dados eficiente para fins estatísticos ou de triagem. Usando fluorescentes padrões talão, o número de proteínas capturados por cada talão pode ser estimada. Muito pouco material de origem é necessária para a captura e detecção, de forma limitada e amostras de analitos escassos ainda podem ser analisados ​​para múltiplas interações. Apesar de IP FCM-não requer engenharia genética, epítopo-tagging, desnaturação, ou in-vitro mistura de proteínas em um ambiente não-fisiológica, pode ser acoplado com estas e outras técnicas, tornando-se um instrumento valioso e acessível, com aplicabilidade a muitos sistemas biológicos. Solução de problemas: Muitos IP-FCM experimentos geram dados de interação de proteínas úteis na primeira vez que são tentados. Entretanto, a otimização da FCM-IP pode melhorar a conjugação de anticorpos para contas, captação de proteínas complexas, e sonda fluorescente vinculativo. A eficiência de conjugação de anticorpos IP pode ser determinada pela sondagem contas acoplado diretamente com um anticorpo anti-imunoglobulina. Se essa eficiência é baixa, aumentando a concentração de anticorpos durante a reação de acoplamento podem permitir mais anticorpos IP para anexar a cada esfera. Isto pode aumentar a capacidade de ligação do lote IP talão, resultando na captura avançada e detecção de analitos. Outros primário-amina contendo moléculas (por exemplo, Tris, albumina de soro bovino) não deve estar presente durante a reação de acoplamento, uma vez que estas possam competir com o mAb para talão de fixação, resultando em acoplamento mAb baixo. Se conjugação de mAb aos grânulos prova problemático por outros motivos, ao invés contas pode ser acoplado a avidina / estreptavidina, e anticorpos biotinilados pode, posteriormente, ser não-covalente e usado para immunprecipitation. Apesar de conjugação de anticorpos boa, a detecção inicial de talão associado fluorescência pode ser baixo. Captura, primeiro complexo em si pode ser baixa. Porque o IP-FCM depende da concentração de analitos, aumentando o número de células lisadas por volume de unidade de lise pode aumentar a captura e detecção do analito. Além disso, a captura pode ser aumentada pela diminuição do número de contas IP incubadas com o lisado, que distribui complexos capturado em contas de menos, e resulta em aumento de fluorescência média por talão quando sondada. Em segundo lugar, é possível que o acesso do anticorpo sonda para os complexos capturada é estericamente impedidas pelo anticorpo immunoprecipitating. Neste caso, o problema pode ser resolvido utilizando anticorpos diferentes para capturar e / ou sonda.

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>carboxylate-modified polystyrene latex (CML) beads (5mm surfactant-free)</td>
<td>Reagent</td>
<td>Interfacial Dynamics Corporation/Molecular Probes</td>
<td>2-5000</td>
<td>Store at 4&deg;C.</td>
</tr>
<tr>
<td>Rainbow Calibration Particles</td>
<td>Reagent</td>
<td>Spherotech, Inc.</td>
<td>RCP-30-5A</td>
<td>Store at 4&deg;C.</td>
</tr>
<tr>
<td>2-[N-Morpholino] ethanesulfonic acid (MES)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>M-5287</td>
<td>&nbsp;</td>
<tr>
<td>1-Ethyl-3-(3-dimethylaminopropl) carbodiimide HCl (EDAC)</td>
<td>Reagent</td>
<td>Pierce Sigma</td>
<td>22980 E-6383</td>
<td>Store at -20&deg;C under desiccating conditions.</td>
</tr>
<tr>
<td>Protease Inhibitor General Use Cocktail, containing AEBSF, Bestatin, Aprotinin, EDTA, E-64, and Leupeptin</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P-2714</td>
<td> Prepare 100x stock solution in H2O. Aliquot and store at -20&deg;C. Handle with gloves.</td>
</tr>
<tr>
<td>PCR plates, 96-well 0.2mL, natural, tall chimney</td>
<td>Reagent</td>
<td>Fisher</td>
<td>08-408-230</td>
<td>For use in place of microcentrifuge tubes during FCM staining</td>
</tr>		</tbody>
		</table>
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ip-fcm:  immunoprecipitation detected by flow cytometry

Imunoprecipitação detectada por citometria de fluxo (IP-FCM) é um método eficiente para a detecção e quantificação de interações proteína-proteína. O princípio básico que se estende de ELISA sanduíche, onde o analito capturado primária pode ser detectado em conjunto com outras moléculas associadas fisicamente dentro de complexos multiprotein. O procedimento envolve o acoplamento covalente de microesferas de látex de poliestireno com immunoprecipitating anticorpos monoclonais (mAb) específico para uma proteína de interesse, incubando estas contas com lisados ​​celulares, sondagem complexos de proteína capturadas com fluorocromo-sondas, e análise de talão associado fluorescência por citometria de fluxo. FCM-IP é extremamente sensível, permite a análise de proteínas em sua nativa (não-desnaturado) do estado, e é passível de qualquer análise semi-quantitativa ou quantitativa. Como vantagens adicionais, IP-FCM não requer engenharia genética ou equipamentos especializados, além de um citômetro de fluxo, e pode ser facilmente adaptado para aplicações de alto rendimento.

Watch this Scientific Journal Video about IP-FCM:  Immunoprecipitation Detected by Flow Cytometry at JoVE.com

IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

O método IP-FCM é apresentado, o que permite uma sensível, avaliação, robusto bioquímica de espécies nativas interações proteína-proteína, sem a necessidade de engenharia genética ou grandes amostras.

FCM-IP: Imunoprecipitação Detectado por citometria de fluxo

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic ...

ip-fcm-immunoprecipitation-detected-by-flow-cytometry

Research

JoVE Journal

Biology

17.4K visualizações.  Mayo Clinic. O método IP-FCM é apresentado, o que permite uma sensível, avaliação, robusto bioquímica de espécies nativas interações proteína-proteína, sem a necessidade de engenharia genética ou grandes amostras.

Vídeo: IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes.
The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body1. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes2,3.
The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies.
Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.

This protocol describes a rapid technique to quantify the translocation of GLUT4 from the cytoplasm to the plasma membrane of cells by flow cytometry.

Medição quantitativa do GLUT4 translocação para a membrana plasmática por citometria de fluxo

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

Biologia

Flow Cytometry Purification of Mouse Meiotic Cells

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 unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

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 ...

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 ...

Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and pathophysiology. This protocol describes a technique where mitochondria isolated from rodent tissue are immunolabeled and analyzed by flow cytometry. Mitochondria are isolated from rodent spinal cords and subjected to a rapid enrichment step so as to remove myelin, a major contaminant of mitochondrial fractions prepared from nervous tissue. Isolated mitochondria are then labeled with an antibody of choice and a fluorescently conjugated secondary antibody. Analysis by flow cytometry verifies the relative purity of mitochondrial preparations by staining with a mitochondrial specific dye, followed by detection and quantification of immunolabeled protein. This technique is rapid, quantifiable and high-throughput, allowing for the analysis of hundreds of thousands of mitochondria per sample. It is applicable to assess novel proteins at the mitochondrial surface under normal physiological conditions as well as the proteins that may become mislocalized to this organelle during pathology. Importantly, this method can be coupled to fluorescent indicator dyes to report on certain activities of mitochondrial subpopulations and is feasible for mitochondria from the central nervous system (brain and spinal cord) as well as liver.

Described here is a method to detect and quantify mitochondrial outer membrane proteins by immunolabeling of mitochondria isolated from rodent tissue and analysis by flow cytometry. This method can be extended to assess functional aspects of mitochondrial subpopulations.

Imunodetecção de membrana externa proteínas por citometria de fluxo de mitocôndrias isoladas

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and ...

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 - 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 ...

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 ...

Measuring Endoreduplication by Flow Cytometry of Isolated Tuber Protoplasts

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated with specialization, development and increase in cellular size. In plants, endoreduplication seems to facilitate the growth and expansion of certain tissues and organs. Among them is the tuber of potato (Solanum tuberosum), which undergoes considerable cellular expansion in fulfilling its function of carbohydrate storage. Thus, endoreduplication may play an important role in how tubers are able to accommodate this abundance of carbon. However, the cellular debris resulting from crude nuclear isolation methods of tubers, methods that can be used effectively with leaves, precludes the estimation of the tuber endoreduplication index (EI). This article presents a technique for assessing tuber endoreduplication through the isolation of protoplasts while demonstrating representative results obtained from different genotypes and compartmentalized tuber tissues. The major limitations of the protocol are the time and reagent costs required for sample preparation as well as relatively short lifespan of samples after lysis of protoplasts. While the protocol is sensitive to technical variation, it represents an improvement over traditional methods of nuclear isolation from these large specialized cells. Possibilities for improvements to the protocol such as recycling enzyme, the use of fixatives, and other alterations are proposed.

The protocol described herein is a method for measuring endoreduplication within tubers of potato (Solanum tuberosum). It includes plasmolysis and protoplast extraction steps to decrease the noise and debris in downstream flow cytometric analysis.

Endoreduplicação por citometria de fluxo de protoplastas de tubérculos isolados de medição

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated ...

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion and the spread of neurodegenerative diseases. The commonly used laboratory strains of the amoeba Dictyostelium discoideum have extremely high fluid uptake rates when grown in nutrient medium, over 90% of which is due to macropinocytosis. In addition, many of the known core components of mammalian macropinocytosis are also present, making it an excellent model system for studying macropinocytosis. Here, the standard technique to measure internalized fluid using fluorescent dextran as a label is adapted to a 96-well plate format, with the samples analyzed by flow cytometry using a high-throughput sampling (HTS) attachment.
Cells are fed non-quenchable fluorescent dextran for a pre-determined length of time, washed by immersion in ice-cold buffer and detached using 5 mM sodium azide, which also stops exocytosis. Cells in each well are then analyzed by flow cytometry. The method can also be adapted to measure membrane uptake and phagocytosis of fluorescent beads or bacteria.
This method was designed to allow measurement of fluid uptake by Dictyostelium in a high-throughput, labor and resource efficient manner. It allows simultaneous comparison of multiple strains (e.g. knockout mutants of a gene) and conditions (e.g. cells in different media or treated with different concentrations of inhibitor) in parallel and simplifies time-courses.

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Macropinocytosis, large-scale non-specific fluid uptake, is important in many areas of clinical biology including immunology, infection, cancer, and neurodegenerative diseases. Here, existing techniques have been adapted to allow high-throughput, single-cell resolution measurement of macropinocytosis in the macropinocytosis model organism Dictyostelium discoideum using flow cytometry.

Medição do elevado-throughput de Dictyostelium discoideum macropinocitose por citometria de fluxo

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion ...

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from low-sensitivity, indirect read-outs, limited throughput, and/or population-level rather than single-cell resolution. We designed a flow cytometry-based single methodology that addresses all of these limitations: Distributed Amphifluoric FRET or DAmFRET. DAmFRET detects and quantifies protein self-assemblies by sensitized emission FRET in vivo, enables deployment across model systems-from yeast to human cells-and achieves sensitive, single-cell, high-throughput read-outs irrespective of protein localization or solubility.

This article describes a FRET-based flow cytometry protocol to quantify protein self-assembly in both S. cerevisiae and HEK293T cells.