Este estudo foi apoiado por um Conselho Ciências Naturais e Engenharia do Canadá (NSERC) e uma bolsa de pesquisa Alberta Agricultura Financiamento Consórcio conceder a DRB. AMR é suportado através de um canadense Vanier de Bolsas de Pós-Graduação NSERC, da Universidade de Alberta estágio de ensino e uma rainha Elizabeth II bolsa de pós-graduação.

Este procedimento pode ser realizado em 500 mL mangueiras siliconizadas ou 6 mL de poliestireno tubos de fundo redondo de FACS. O último é normalmente utilizado na realização de análise de viabilidade em conjunto com outros procedimentos. Todos os volumes indicados são para 6 mL de poliestireno tubos de fundo redondo de FACS. Para 500 mL mangueiras siliconizadas, de reduzir todos os volumes por 05/01. A concentração ótima para a análise de citometria de fluxo é de 2-4 x 10 6 células por volume de 200 mL. Perda de células pode resultar desse procedimento e, portanto, recomendamos que cada amostra composta de 4 x 10 6 células no início do procedimento. 1. Preparação de células <ol><li> Células colheita - referem-se a procedimentos específicos para linhagens de células correspondente ou isolamentos de células primárias. </li><li> Centrifugar as amostras a 335 x g por 10 minutos e decantar o sobrenadante. </li><li> Ressuspender as células em 2 mL de fosfato x 1 solução salina tamponada (PBS) - / - (sem cálcio, magnésio não). </li><li> Centrifugar as amostras a 335 x g por 10 minutos e decantar o sobrenadante. </li><li> Ressuspender as células em 1 mL 1 x anexina V buffer de ligação. </li><li> Centrifugar as amostras a 335 x g por 10 minutos e decantar o sobrenadante. </li><li> Ressuspender as células em 100 mL 1 x anexina V buffer de ligação. </li></ol> 2. Aplicação de anexina V / PI Stain <ol><li> Adicionar anexina V de acordo com as recomendações do fabricante [por exemplo, 5 mL anexina V Alexa Fluor 488 (Molecular Probes, A13201)]. </li><li> Incubar os tubos no escuro por 15 minutos em temperatura ambiente. </li><li> Adicionar 100 mL de tampão 1 x V anexina ligação a cada tubo de reacção. Deve haver cerca de 200 mL em cada tubo. </li><li> Adicionar 4 mL de PI (Sigma, Cat # P-4864-10ML) que foi diluída 1:10 em tampão 1 x V anexina vinculativo (isto é, PI 1 mL com 9 x 1 mL de buffer V anexina ligação). O resultado será uma concentração final de 2 PI mcg / mL em cada amostra. </li><li> Incubar os tubos no escuro por 15 minutos em temperatura ambiente. </li><li> Adicionar 500 mL 1 x anexina V tampão de ligação para lavar as células. </li><li> Centrifugar as amostras a 335 x g por 10 minutos e decantar o sobrenadante. </li><li> Ressuspender as células em 500 mL de buffer 1 x aderência à anexina V e 500 mL de formaldeído a 2% para criar uma solução a 1% de formaldeído (fixador). Mix tubos sacudindo gentil. </li><li> Fix amostras no gelo por 10 minutos. Alternativamente, as amostras podem ser armazenadas durante a noite a 4 ° C no escuro. Se o último método é escolhido, certifique-se de ser coerente em todos os tubos rotulados com anexina V / PI (incluindo controles de compensação). </li><li> Adicionar 1 mL 1 x PBS - / - para cada amostra e misturar delicadamente por flicking. </li><li> Centrifugar os tubos a 425 x g por oito minutos e decantar o sobrenadante. </li><li> Repita os passos 2.10 e 2.11. </li><li> Ressuspender pellet sacudindo o tubo. </li><li> Adicionar 16 mL de 1:100 diluídos RNase A (Sigma, R4642) para dar uma concentração final de 50 mcg / mL. Incubar por 15 min a 37 ° C. </li><li> Adicionar 1 mL 1 x PBS - / - e misture delicadamente sacudindo. </li><li> Centrifugar os tubos a 425 x g por oito minutos. </li><li> Amostras está agora pronto para ser analisado. Alternativamente, as amostras podem ser utilizados para as etapas subseqüentes coloração se anexina / PI coloração está sendo realizada em paralelo com outros procedimentos. </li></ol> 3. Resultados representativos: Exemplos de tipos de células e linhas de células que têm diferentes graus de falso-positivo coloração PI são mostrados na Figura 1. Para explicar os eventos falso-positivos, as células foram fixadas e então tratadas com RNase A. Isto resulta em uma diminuição significativa no número de falso-positivo de eventos, em comparação com as células que não foram submetidos a tratamento RNase (Figura 2B). Figura 2C mostra imagens representativas das células que foram submetidos a RNase tratamento, em comparação com as células que não tinha tratamento RNase. A Figura 3 mostra como a modificação anexina V / PI protocolo, com fixação e etapas de tratamento RNase, melhora a protocolos convencionais, limitando o número de eventos de falso-positivo coloração. <img src="/files/ftp_upload/2597/2597fig1.jpg" alt="Figura 1" /> Figura 1. Convencionais propidium protocolos de coloração iodeto de levar a um número significativo de falsos acontecimentos positivos. Temos previamente avaliados extensão da coloração falso positivo em células primárias em uma ampla gama de modelos animais, bem como em uma variedade de linhas de células 10. Imagens representativas mostram extensão da coloração falso positivo em três populações de células únicas (uma linhagem de células comumente usados ​​- macrófagos RAW e duas pilhas - macrófagos da medula óssea de camundongos (BMM) e macrófagos goldfish primário renal (PKM)). Verdadeiros acontecimentos positivos exibir o esperado co-localização entre PI e DRAQ5 no interior do compartimento nuclear (áreas amarelas representam co-localização entre PI e DRAQ5). DRAQ5 é altamente membrane permeável corante que precisa compartimento manchas nuclear em ambas as células vivas e mortas 10,11,12. Para facilitar a determinação visual da co-localização DRAQ5 mancha foi dado um pseudocolour verde. <img src="/files/ftp_upload/2597/2597fig2.jpg" alt="Figura 2" /> Figura 2. Melhorar a precisão do PI coloração nuclear. (A) Avaliamos a precisão de PI coloração nuclear com base no grau de semelhança de uma série de bem caracterizados manchas nuclear (BrdU, 7-AAD e DAPI) para DRAQ5. BrdU é um nucleosídeo sintético que é incorporada ao DNA de células em proliferação, e como tal só mancha no interior do compartimento nuclear. 7-AAD tem uma alta afinidade por DNA e, semelhante ao PI, não pode atravessar uma membrana plasmática intacta. DAPI também tem uma forte afinidade por DNA e é o mais comumente usado nuclear mancha em microscopia fluorescente. O grau de similaridade foi&gt; 99% entre BrdU, 7-AAD, DAPI e DRAQ5, destacando a sua capacidade de precisão mancha do núcleo da célula em RAW 264,7 macrófagos. Em contraste, coloração citoplasmática PI baseado em protocolos convencionais leva a uma diminuição acentuada na similaridade de coloração por cento em relação ao DRAQ5. (B) Resultados semelhantes foram observados em duas pilhas testadas: os macrófagos da medula óssea de camundongos (BMM) e macrófagos goldfish primário renal (PKM). Incorporação de 50 mg / mL de RNase A na fase específica dentro do processo de coloração não remove a coloração nuclear PI e aumenta significativamente o grau de semelhança em relação ao DRAQ5 coloração. (C) Imagens representativas de macrófagos primárias de rim de mostrar o grau de semelhança para as células com e sem tratamento RNase. Para facilitar a determinação visual das diferenças entre os tratamentos, DRAQ5 foi dado um verde. Áreas amarelas representam co-localização entre BrdU e DRAQ5 e PI e DRAQ5. <img src="/files/ftp_upload/2597/2597fig3.jpg" alt="Figura 3" /> Figura 3. Modificado anexina V / PI reduz significativamente a apoptose false / eventos necrótico. Macrófagos goldfish primário renal (PKM) foram coradas com convencional e modificado anexina V / PI protocolos. Gráficos de dispersão retratam a anexina V / PI mancha em PKM goldfish. A dispersão da esquerda mostra convencionais anexina V coloração / PI (sem RNase) de células PKM. A dispersão da direita mostra células PKM manchadas com o anexina V modificado / protocolo IP (com RNase). A adição dos resultados da RNase em uma acentuada redução do PI coloração devido à remoção de manchas falsos positivos. PKM células foram então analisados ​​com base em populações distintas dentro da progenitores culturas precoce (EP), monócitos (Mono) e macrófagos maduros (Mat mac). A redução do número de eventos PI positivo, devido à remoção de falsos eventos positivos é mais pronunciado em grandes macrófagos maduros do que em menor células progenitoras cedo. Conclusões baseadas em protocolos convencionais teria erroneamente sugere uma correlação positiva na morte celular para subconjuntos de macrófagos mais maduro.

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	<li>Vermes, I., Haanen, C., Steffens-Nakken, H., Reutelingsperger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+assay+for+apoptosis.+Flow+cytometric+detection+of+phosphatidylserine+expression+on+early+apoptotic+cells+using+fluorescein+labeled+Annexin+V.">A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labeled Annexin V.</a> J Immunol Methods. 184, 39-51 (1995).</li><li>Vermes, I., Haanen, C., Reutelingsperger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+of+apoptotic+cell+death.">Flow cytometry of apoptotic cell death.</a> J Immunol Methods. 243, 167-190 (2000).</li><li>Cornelissen, M., Philippe, J., De Sitter, S., De Ridder, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Annexin+V+expression+in+apoptotic+peripheral+blood+lymphocytes:+An+electron+microscopic+evaluation.">Annexin V expression in apoptotic peripheral blood lymphocytes: An electron microscopic evaluation.</a> Apoptosis. 7, 41-47 (2002).</li><li>Fried, J., Perez, A. G., Clarkson, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytofluorometric+analysis+of+cell+cycle+distributions+using+propidium+iodide.+Properties+of+the+method+and+mathematical+analysis+of+the+data.">Flow cytofluorometric analysis of cell cycle distributions using propidium iodide. Properties of the method and mathematical analysis of the data.</a> J Cell Biol. 71, 172-181 (1976).</li><li>Bacso, Z., Everson, R. B., Eliason, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+DNA+of+Annexin+V-binding+apoptotic+cells+is+highly+fragmented.">The DNA of Annexin V-binding apoptotic cells is highly fragmented.</a> Cancer Res. 60, 4623-4628 (2000).</li><li>Darzynkiewicz, Z., Bruno, S., Del Bino, G., Gorczyca, W., Hotz, M. A., Lassota, P., Traganos, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Features+of+apoptotic+cells+measured+by+flow+cytometry.">Features of apoptotic cells measured by flow cytometry.</a> Cytometry. 13, 795-808 (1992).</li><li>Kroemer, G., Dallaporta, B., Resche-Rigon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mitochondrial+death/life+regulator+in+apoptosis+and+necrosis.">The mitochondrial death/life regulator in apoptosis and necrosis.</a> Annu. Rev. Physiol. 60, 619-642 (1998).</li><li>Denecker, G., Vercammen, D., Declercq, W., Vandenabeele, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptotic+and+necrotic+cell+death+induced+by+death+domain+receptors.">Apoptotic and necrotic cell death induced by death domain receptors.</a> Cell Mol. Life Sci. 58, 356-370 (2001).</li><li>Faleiro, L., Lazebnik, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspases+disrupt+the+nuclear-cytoplasmic+barrier.">Caspases disrupt the nuclear-cytoplasmic barrier.</a> J Cell Biol. 151, 951-959 (2000).</li><li>Rieger, A. M., Hall, B. E., Luong, L. T., Schang, L. M., Barreda, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conventional+apoptosis+assays+using+propidium+iodide+generate+a+significant+number+of+false+positives+that+prevent+accurate+assessment+of+cell+death.">Conventional apoptosis assays using propidium iodide generate a significant number of false positives that prevent accurate assessment of cell death.</a> J Immunol Methods. 358, 81-92 (2010).</li><li>Edward, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minireview:+Use+of+DNA-specific+anthraquinone+dyes+to+directly+reveal+cytoplasmic+and+nuclear+boundaries+in+live+and+fixed+cells.">Minireview: Use of DNA-specific anthraquinone dyes to directly reveal cytoplasmic and nuclear boundaries in live and fixed cells.</a> Mol. Cells. 27, 391-396 (2009).</li><li>Njoh, K. L., Patterson, L. 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Não há conflitos de interesse declarados.

A anexina V / PI protocolo aqui apresentado é uma versão modificada de protocolos convencionais e leva em conta a presença de RNA no citoplasma que também tem alta afinidade para o PI. Introdução de RNase A (50 mg / mL), após um passo de 1% de formaldeído de fixação no final do processo de coloração melhora significativamente a precisão da coloração PI nuclear. Sem efeitos negativos são observados na coloração PI nuclear ou membrana plasmática anexina V coloração. Sem tratamento RNase A, até 40% dos resultados PI mancha pode levar a falsos eventos positivos, o que leva a um impacto potencialmente significativo e negativo sobre as conclusões a jusante 10. Nosso modificada anexina V protocolo de coloração / PI é simples e tem sido utilizada de forma eficaz em uma ampla gama de tipos de células (células primário: os macrófagos da medula óssea de camundongos e esplenócitos, células residentes suína pulmão, macrófagos alveolares do pulmão, os isolados do nó de linfa mesentérica, leucócitos do sangue periférico , esplenócitos, células blastoderma frango; macrófagos goldfish primárias de rim; leucócitos do sangue periférico de carpa; linhas Cell: RAW 264,7 macrófagos, células Jurkat T, suína 3D4/31 macrófagos, CCL-71 fibroblastos fin goldfish, peixe-gato 3B11 células B 10). É importante notar que as células são diferencialmente afetados pela coloração PI falso positivo, com pilhas geralmente tendo um maior número de falsos eventos positivos 10. As diferenças de coloração PI falso positivo entre essas populações celulares pode ser parcialmente atribuída a diferenças no tamanho das células, mas também podem resultar de diferenças no conteúdo de RNA. Como a incorporação, tais deste procedimento é particularmente relevante para sistemas experimentais que utilizam, entre outras, células submetidos a estresse genotóxico, as células tratadas com drogas parada do ciclo celular, tais como timidina ou hidroxiuréia, as células infectadas por vírus, e estudos sobre células embrionárias, onde a progressão do desenvolvimento é caracterizado por mudanças discretas na síntese de RNA celular.

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<td>1 x PBS-/-</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1 x Annexin V Binding Buffer</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>556454</td>
<td>&nbsp;</td>
<tr>
<td>Annexin V-Alexa Fluor 488</td>
<td>&nbsp;</td>
<td>Molecular Probes (Invitrogen)</td>
<td>A13201</td>
<td>&nbsp;</td>
<tr>
<td>Propidium iodide (PI)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P4864</td>
<td>1 mg/mL in H2O</td>
</tr>
<tr>
<td>2% Formaldehyde</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>F1268</td>
<td>&nbsp;</td>
<tr>
<td>RNase A from bovine pancreas</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>R4642</td>
<td>&nbsp;</td>
<tr>
<td>DRAQ5</td>
<td>&nbsp;</td>
<td>Biostatus</td>
<td>DR50050</td>
<td>Dilute 1:60 in 1 x PBS-/-</td>
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modified annexin v/propidium iodide apoptosis assay for accurate assessment of cell death

Estudos da apoptose celular foram afetados significativamente desde a introdução da citometria de fluxo baseado em métodos. Iodeto de propídio (PI) é amplamente usado em conjunto com anexina V para determinar se as células são viáveis, apoptóticas ou necróticas através de diferenças na integridade da membrana plasmática e 1,2 permeabilidade. A anexina V / PI protocolo é um método comumente usado para estudar as células apoptóticas 3. PI é utilizado mais frequentemente do que outras manchas nuclear porque é econômico, estável e um bom indicador de viabilidade celular, com base em sua capacidade de excluir corante em células vivas 4,5. A capacidade de PI para inserir uma célula é dependente da permeabilidade da membrana; PI não mancha ao vivo ou no início de células apoptóticas devido à presença de uma membrana plasmática intacta 1,2,6. No final de células apoptóticas e necróticas, a integridade das membranas plasmática e nuclear diminui 7,8, permitindo PI para passar através das membranas, intercalam em ácidos nucléicos, e apresentar fluorescência vermelha 1,2,9. Infelizmente, descobrimos que convencionais anexina V / PI protocolos levar a um número significativo de falsos eventos positivos (até 40%), que estão associados a PI de coloração de RNA no compartimento citoplasmático 10. Pilhas e linhas celulares em uma ampla gama de modelos animais são afetados, com células grandes (nuclear: relações citoplasmática &lt;0,5), mostrando a maior ocorrência 10. Aqui, nós demonstramos um método modificado anexina V / PI que proporciona uma melhoria significativa para a avaliação da morte celular em comparação com métodos convencionais. Este protocolo tira proveito das mudanças na permeabilidade celular durante célula de fixação para promover a entrada de RNase A para as células seguintes coloração. Tanto tempo e concentração de RNase A foram otimizados para a remoção de RNA citoplasmático. O resultado é uma melhoria significativa em relação convencional anexina V / PI protocolos (&lt;5% eventos com coloração citoplasmática PI).

Watch this Scientific Journal Video about Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death at JoVE.com

Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death

Um método preciso para a avaliação da morte celular é descrito. O protocolo convencional melhora a anexina V / iodeto de propídio (PI) protocolos, que apresentam até 40% de falsos-positivos eventos em linhagens celulares e pilhas de uma ampla gama de modelos animais.

Modificado anexina V / propídio Assay Apoptose Iodeto para avaliação rigorosa da morte celular

Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely ...

modified-annexin-vpropidium-iodide-apoptosis-assay-for-accurate

Research

JoVE Journal

Biology

102.4K visualizações.  University of Alberta. Um método preciso para a avaliação da morte celular é descrito. O protocolo convencional melhora a anexina V / iodeto de propídio (PI) protocolos, que apresentam até 40% de falsos-positivos eventos em linhagens celulares e pilhas de uma ampla gama de modelos animais.

Vídeo: Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death

Modified Mouse Embryonic Stem Cell based Assay for Quantifying  Cardiogenic Induction Efficiency

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways. One of the hurdles to its understanding has been the varied methods in correlating changes of key signaling events to differentiation efficiency. We describe here the use of a mouse embryonic stem (ES) cell based assay to identify critical time windows for Wnt/&beta;-catenin and BMP signal activation during cardiogenic induction. By scoring for contracting embryonic bodies (EBs) in a 96-well plate format, we can quickly quantify cardiogenic efficiency and identify crucial time windows for Wnt/&beta;-catenin and BMP signal activation in a time course following specific modulator treatments. The principal outlined here is not limited to cardiac induction alone, and can be applied towards the study of many other cell lineages. In addition, the 96-well format has the potential to be further developed as a high throughput, automated assay to allow for the testing of more sophisticated experimental hypotheses.

We describe the use of a mouse ES cell based assay to identify critical time windows for Wnt/&#946;-catenin and BMP signal activation during cardiogenic induction. The method provides a standardized platform that reliably quantifies cardiogenic efficiency, and it is applicable to the study of other cell lineages.

Modificado embrionárias de camundongos com células-tronco Ensaio baseado para quantificar a eficiência de indução cardiogênico

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways.  One of the hurdles ...

Biologia

A &#946;-glucuronidase (GUS) Based Cell Death Assay

We have developed a novel transient plant expression system that simultaneously expresses the reporter gene, &beta;-glucuronidase (GUS), with putative positive or negative regulators of cell death. In this system, N. benthamiana leaves are co-infiltrated with a 35S driven expression cassette containing the gene to be analyzed, and the GUS vector pCAMBIA 2301 using Agrobacterium strain LBA4404 as a vehicle. Because live cells are required for GUS expression to occur, loss of GUS activity is expected when this marker gene is co-expressed with positive regulators of cell death. Equally, increased GUS activity is observed when anti-apoptotic genes are used compared to the vector control. As shown below, we have successfully used this system in our lab to analyze both pro- and anti-death players. These include the plant anti-apoptotic Bcl-2 Associated athanoGene (BAG) family, as well as, known mammalian inducers of cell death, such as BAX. Additionally, we have used this system to analyze the death function of specific truncations within proteins, which could provide clues on the possible post-translational modification/activation of these proteins. Here, we present a rapid and sensitive plant based method, as an initial step in investigating the death function of specific genes.

A &amp;#946;-glucuronidase GUS Based Cell Death Assay

Programmed cell death assays commonly used in mammalian systems such as DNA laddering or TUNEL assays, are often difficult to reproduce in plants. In combination with a GUS reporter system, we propose a rapid, plant based transient assay to analyze the potential death properties of specific genes.

A β-glucuronidase (GUS) Ensaio Baseado Morte celular

We have developed a novel transient plant expression system that simultaneously expresses the reporter gene, &beta;-glucuronidase (GUS), with putative ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

Ativação da apoptose por microinjeção citoplasmática de citocromo C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and organs1,2,6,7. During development, toxic chemicals or genetic alterations can cause an increase in PCD or change PCD patterns resulting in developmental abnormalities and birth defects3-5. To understand the etiology of these defects, the study of embryos can be complemented with in vitro assays that use differentiating embryonic stem (ES) cells.
Apoptosis is a well-studied form of PCD that involves both intrinsic and extrinsic signaling to activate the caspase enzyme cascade. Characteristic cell changes include membrane blebbing, nuclear shrinking, and DNA fragmentation. Other forms of PCD do not involve caspase activation and may be the end-result of prolonged autophagy. Regardless of the PCD pathway, dying cells need to be removed. In adults, the immune cells perform this function, while in embryos, where the immune system has not yet developed, removal occurs by an alternative mechanism. This mechanism involves neighboring cells (called "non-professional phagocytes") taking on a phagocytic role-they recognize the 'eat me' signal on the surface of the dying cell and engulf it8-10. After engulfment, the debris is brought to the lysosome for degradation. Thus regardless of PCD mechanism, an increase in lysosomal activity can be correlated with increased cell death.
To study PCD, a simple assay to visualize lysosomes in thick tissues and multilayer differentiating cultures can be useful. LysoTracker dye is a highly soluble small molecule that is retained in acidic subcellular compartments such as the lysosome11-13. The dye is taken up by diffusion and through the circulation. Since penetration is not a hindrance, visualization of PCD in thick tissues and multi-layer cultures is possible12,13. In contrast, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) analysis14, is limited to small samples, histological sections, and monolayer cultures because the procedure requires the entry/permeability of a terminal transferase.
In contrast to Aniline blue, which diffuses and is dissolved by solvents, LysoTracker Red DND-99 is fixable, bright, and stable. Staining can be visualized with standard fluorescent or confocal microscopy in whole-mount or section using aqueous or solvent-based mounting media12,13. Here we describe protocols using this dye to look at PCD in normal and sonic hedgehog null mouse embryos. In addition, we demonstrate analysis of PCD in differentiating ES cell cultures and present a simple quantification method. In summary, LysoTracker staining can be a great complement to other methods of detecting PCD.

We present a simple protocol to visualize regions of programmed cell death (PCD) in mouse embryos and differentiating embryonic stem (ES) cell cultures using a highly soluble dye called LysoTracker.

Uso de Lyso Tracker para detectar a morte celular programada em embriões e diferenciação das células-tronco embrionárias

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

O tomate / GFP-FLP / FRT Method for Imaging ao vivo de Mosaico Adulto<em&gt; Drosophila</em&gt; As células fotorreceptoras

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important checkpoints of apoptosis, including mitochondrial fragmentation, release of mitochondrial cytochrome c into the cytosol, activation of caspases, chromatin condensation, DNA damage, nuclear fragmentation, plasma membrane blebbing, cell shrinkage, cell surface exposure of phosphatidylserine, and formation of apoptotic bodies. Anastasis can occur when apoptotic stimuli are removed prior to death, thereby allowing dying cells to reverse apoptosis and potentially other death mechanisms. Therefore, anastasis appears to involve physiological healing processes that could also sustain damaged cells inappropriately. The functions and mechanisms of anastasis are still unclear, hampered in part by the limited tools for detecting past events after the recovery of apparently healthy cells. Strategies to detect anastasis will enable studies of the physiological mechanisms, the hazards of undead cells in disease pathology, and potential therapeutics to modulate anastasis. Here, we describe effective strategies using live cell microscopy and a mammalian caspase biosensor&#160;for identifying and tracking anastasis in mammalian cells.

The term anastasis refers to the phenomenon in which dying cells reverse a cell suicide process at a late stage, repair themselves, and ultimately survive. Here we demonstrate protocols for detecting and tracking cells that undergo anastasis.

Estratégias para Rastreamento Anastasis, uma célula de sobrevivência Fenômeno que reverte apoptose

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

Avaliação da Seletiva mRNA Tradução em células de mamíferos por polissoma Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...

An Iodide-Yellow Fluorescent Protein-Gap Junction-Intercellular Communication Assay

Gap junctions (GJs) are cell membrane channels that allow diffusion of molecules smaller than 1 kDa between adjacent cells. As they have physiological and pathological roles, there is need of high-throughput screening (HTS) assays to identify GJ modulators in drug discovery and toxicology assays. A novel iodide-yellow fluorescent protein-gap junction-intercellular communication (I-YFP-GJIC) assay fulfills this need. It is a cell-based assay including acceptor and donor cells that are engineered to stably express a yellow fluorescent protein (YFP) variant, whose fluorescence is sensitively quenched by iodide, or SLC26A4, an iodide transporter, respectively. When iodide is added to a mixed culture of the two cell types, they enter the donor cells via the SLC26A4 transporter and diffuse to the adjacent acceptor cells via GJs where they quench the YFP fluorescence. YFP fluorescence is measured well by well in a kinetic mode. The YFP quenching rate reflects GJ activity. The assay is reliable and rapid enough to be used for HTS. The protocol for the I-YFP-GJIC assay using the LN215 cells, human glioma cells, is described.

Here, we present a protocol for a novel gap junction intercellular communication assay designed for the high-throughput screening of gap junction-modulating chemicals for drug discovery and toxicological assessment.

Um ensaio de comunicação intercelular junção iodeto-amarelo fluorescente proteína-Gap

Gap junctions (GJs) are cell membrane channels that allow diffusion of molecules smaller than 1 kDa between adjacent cells. As they have physiological and ...

Single Myofiber Culture Assay for the Assessment of Adult Muscle Stem Cell Functionality Ex Vivo

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells leave their quiescent state and activate the myogenic program ultimately leading to the repair of damaged tissue concomitant with the replenishment of the muscle stem cell pool. Various factors influence muscle stem cell activity, among them intrinsic stimuli but also signals from the direct muscle stem cell environment, the stem cell niche. The isolation and culture of single myofibers with their associated muscle stem cells preserves most of the interaction of the stem cell with its niche and is, therefore, the closest possibility to study muscle stem cell functionality ex vivo. Here, a protocol for the isolation, culture, siRNA transfection and immunostaining of muscle stem cells on their respective myofibers from mouse EDL (extensor digitorum longus) muscles is provided. The experimental conditions outlined here allow the study and manipulation of muscle stem cells ex vivo including investigation of myogenic activity without the inherent need for in vivo animal experiments.

In this protocol an in vitro culturing and functional analysis method for muscle stem cells is described, which preserves most of their interactions with their endogenous niche.

Ensaio de cultura de myofibra única para a avaliação da funcionalidade de células-tronco musculares adultas Ex Vivo

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells ...

Neutrophil Lifespan Extension with CLON-G and an In Vitro Spontaneous Death Assay

The average lifespan of a neutrophil is less than 24 h, which limits basic research on neutrophils and the application of neutrophil studies. Our previous research indicated that multiple pathways could mediate the spontaneous death of neutrophils. A cocktail was developed by simultaneously targeting these pathways, caspases-lysosomal membrane permeabilization-oxidant-necroptosis inhibition plus granulocyte colony-stimulating factor (CLON-G), which prolonged the neutrophil lifespan to greater than 5 days without significantly compromising the neutrophil function. Concurrently, a reliable and stable protocol for assessing and evaluating neutrophil death was also developed. In this work, we show that CLON-G can prolong the neutrophil lifespan in vitro to more than 5 days, and we exhibit the lengthening of the neutrophil lifespan with FACS and confocal fluorescence microscopy. This report introduces procedures for the preparation of CLON-G and showcases an in vitro spontaneous death assay of neutrophils, which can be used for the study of neutrophils and for subsequently interrogating neutrophil death, thus providing a reliable resource for the neutrophil community.

Neutrophil Lifespan Extension with CLON-G and an In Vitro Spontaneous Death Assay

This protocol details the preparation of CLON-G to extend the neutrophil lifespan to greater than 5 days and provides a reliable procedure for evaluating neutrophil death with flow cytometry and confocal fluorescence microscopy.

Extensão da vida útil dos neutrófilos com CLON-G e ensaio de morte espontânea in vitro

The average lifespan of a neutrophil is less than 24 h, which limits basic research on neutrophils and the application of neutrophil studies. Our previous ...