Name: modified annexin v/propidium iodide apoptosis assay for accurate assessment of cell death
Uploaded: 2011-04-24T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death at JoVE.com

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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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.

<table><tbody><tr><td>1 x PBS-/-</td><td></td><td></td><td></td></tr><tr><td>1 x Annexin V Binding Buffer</td><td>BD Biosciences</td><td>556454</td><td></td></tr><tr><td>Annexin V-Alexa Fluor 488</td><td>Molecular Probes, Life Technologies</td><td>A13201</td><td></td></tr><tr><td>Propidium iodide (PI)</td><td>Sigma-Aldrich</td><td>P4864</td><td>1 mg/mL in H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>2% Formaldehyde</td><td>Sigma-Aldrich</td><td>F1268</td><td></td></tr><tr><td>RNase A from bovine pancreas</td><td>Sigma-Aldrich</td><td>R4642</td><td></td></tr><tr><td>DRAQ5</td><td>Biostatus</td><td>DR50050</td><td>Dilute 1:60 in 1 x PBS-/-</td></tr></tbody></table>

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

Nanyang Technological University

Research

JoVE Journal

Biology

103.1K 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

Detection and Isolation of Apoptotic Bodies to High Purity

Apoptotic bodies (ApoBDs), microvesicles and exosomes are the key members of the extracellular vesicle family, with ApoBDs being one of the largest type. It has been proposed that ApoBDs can aid cell clearance as well as intercellular communication through trafficking biomolecules. Conventional approaches used for the identification and isolation of ApoBDs are often limited by the lack of accurate quantification and low sample purity. Here, we describe a workflow to confirm the induction of apoptosis, validate ApoBD formation, and isolate ApoBDs to high purity. We will also outline and compare fluorescence-activated cell sorting (FACS) and differential centrifugation based approaches to isolate ApoBDs. Furthermore, the purity of isolated ApoBDs will be confirmed using a previously establish flow cytometry-based staining and analytical method. Taken together, using the described approach, THP-1 monocyte apoptosis and apoptotic cell disassembly was induced and validated, and ApoBD generated from THP-1 monocytes were isolated to a purity of 97-99%.

A workflow using flow cytometry or differential centrifugation is developed to detect, quantify and isolate apoptotic bodies from an apoptotic sample to high purity.

Detecção e isolamento de corpos Apoptotic de alta pureza

Apoptotic bodies (ApoBDs), microvesicles and exosomes are the key members of the extracellular vesicle family, with ApoBDs being one of the largest type. ...

Bioquímica

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depolarization

The central nervous system can experience a number of stresses and neurological insults, which can have numerous adverse effects that ultimately lead to a reduction in neuronal population and function. Damaged axons can release excitatory molecules including potassium or glutamate into the extracellular matrix, which in turn, can produce further insult and injury to the supporting glial cells including astrocytes and oligodendrocytes 8, 16. If the insult persists, cells will undergo programmed cell death (apoptosis), which is regulated and activated by a number of well-established signal transduction cascades 14. Apoptosis and tissue necrosis can occur after traumatic brain injury, cerebral ischemia, and seizures. A classical example of apoptotic regulation is the family of cysteine-dependent aspartate-directed proteases, or caspases. Activated proteases including caspases have also been implicated in cell death in response to chronic neurodegenerative diseases including Alzheimer's, Huntington's, and Multiple Sclerosis 4, 14, 3, 11, 7.
In this protocol we describe the use of the NucView 488 caspase-3 substrate to measure the rate of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte (OLG) cell cultures 15, 5, following exposure to different extracellular stresses such as high concentrations of potassium or glutamate. The conditionally-immortalized N19-OLG cell line (representing the O2A progenitor) was obtained from Dr. Anthony Campagnoni (UCLA Semel Institute for Neuroscience) 15, 5, and has been previously used to study molecular mechanisms of myelin gene expression and signal transduction leading to OLG differentiation (e.g.6, 10). We have found this cell line to be robust with respect to transfection with exogenous myelin basic protein (MBP) constructs fused to either RFP or GFP (red or green fluorescent protein) 13, 12. Here, the N19-OLG cell cultures were treated with either 80 mM potassium chloride or 100 mM sodium glutamate to mimic axonal leakage into the extracellular matrix to induce apoptosis 9. We used a bi-functional caspase-3 substrate containing a DEVD (Asp-Glu-Val-Asp) caspase-3 recognition subunit and a DNA-binding dye 2. The substrate quickly enters the cytoplasm where it is cleaved by intracellular caspase-3. The dye, NucView 488 is released and enters the cell nucleus where it binds DNA and fluoresces green at 488 nm, signaling apoptosis. Use of the NucView 488 caspase-3 substrate allows for live-cell imaging in real-time 1, 10. In this video, we also describe the culturing and transfection of immortalized N19-OLG cells, as well as live-cell imaging techniques.

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depola

Live-cell imaging of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte cell cultures using the NucView 488 caspase-3 substrate. This technique is applicable for programmed cell death assays in real-time in a variety of cell types and tissues.

Monitoramento de Atividades de caspase-3 Clivadas e apoptose das células oligodendrogliais Imortalizado usando Live-célula Imaging and Cleaveable Fluorogênico-dye seguintes substratos de potássio induzida por despolarização da membrana

The central nervous system can experience a number of stresses and neurological insults, which can have numerous adverse effects that ultimately lead to a ...

Neurociência

An Automated Differential Nuclear Staining Assay for Accurate Determination of Mitocan Cytotoxicity

The contribution of mitochondria to oncogenic transformation is a subject of wide interest and active study. As the field of cancer metabolism becomes more complex, the goal of targeting mitochondria using various compounds that inflict mitochondrial damage (so-called mitocans) is becoming quite popular. Unfortunately, many existing cytotoxicity assays, such as those based on tetrazolium salts or resazurin require functional mitochondrial enzymes for their performance. The damage inflicted by compounds that target mitochondria often compromises the accuracy of these assays. Here, we describe a modified protocol based on differential staining with two fluorescent dyes, one of which is cell-permeant (Hoechst 33342) and the other of which is not (propidium iodide). The difference in staining allows living and dead cells to be discriminated. The assay is amenable to automated microscopy and image analysis, which increases throughput and reduces bias. This also allows the assay to be used in high-throughput fashion using 96-well plates, making it a viable option for drug discovery efforts, particularly when the drugs in question have some level of mitotoxicity. Importantly, results obtained by Hoechst/PI staining assay show increased consistency, both with trypan blue exclusion results and between biological replicates when the assay is compared to other methods.

The protocol describes a rapid, high-throughput, reliable, inexpensive, and unbiased assay for efficiently determining cellular viability. This assay is particularly useful when cells&#39; mitochondria have been damaged, which interferes with other assays. The assay uses automated counting of cells stained with two nuclear dyes &#8211; Hoechst 33342 and propidium iodide.

Um ensaio automatizado de coloração nuclear diferencial para determinação precisa da citotoxicidade mitocana

The contribution of mitochondria to oncogenic transformation is a subject of wide interest and active study. As the field of cancer metabolism becomes ...

Pesquisa do câncer

Flow Cytometric Analysis of Apoptotic Biomarkers in Actinomycin D-Treated SiHa Cervical Cancer Cells

Apoptosis biomarkers were investigated in actinomycin D-treated SiHa cervical cancer cells using a benchtop flow cytometer. Early biomarkers (Annexin V and mitochondrial membrane potential) and late biomarkers (caspases 3 and 7, and DNA damage) of apoptosis were measured in experimental and control cultures. Cultures were incubated for 24 hours in a humidified incubator at 37 &#176;C with 5% CO2. The cells were then detached using trypsin and enumerated using a flow cytometric cell count assay. Cells were further analyzed for apoptosis using an Annexin V assay, a mitochondrial electrochemical transmembrane potential assay, a caspase 3/7 assay, and a DNA damage assay. This article provides an overview of apoptosis and traditional flow cytometry, and elaborates flow cytometric protocols for processing and analyzing SiHa cells. The results describe positive, negative, and sub-optimal experimental data. Also discussed are interpretation and caveats in performing flow cytometric analysis of apoptosis using this analytical platform. Flow cytometric analysis provides an accurate measurement of early and late biomarkers for apoptosis.

Apoptosis can be characterized by flow cytometric analysis of early and late apoptotic biomarkers. The cervical cancer cell line, SiHa, was analyzed for apoptosis biomarkers after treatment with Actinomycin D using a benchtop flow cytometer.

Análise por citometria de fluxo de biomarcadores apoptóticos em células de câncer cervical SiHa tratadas com Actinomicina D

Apoptosis biomarkers were investigated in actinomycin D-treated SiHa cervical cancer cells using a benchtop flow cytometer. Early biomarkers (Annexin V ...

Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and decreases waste in costs and reagents. Here we describe optimization of a multiplex assay to assess apoptosis following a palmitic acid (PA) challenge in an in vitro hypothalamic model, using both fluorescent and luminescent based assays to measure viable cell counts and caspase-3/7 activity in a 96-well microtiter plate format. Following PA challenge, viable cells were determined by a resazurin-based fluorescent assay. Caspase-3/7 activity was then determined using a luminogenic substrate, DEVD, and normalized to cell number. This multiplexing assay is a useful technique for determining change in caspase activity following an apoptotic stimulus, such as saturated fatty acid challenge. The saturated fatty acid PA can increase hypothalamic oxidative stress and apoptosis, indicating the potential importance of assays such as that described here in studying the relationship between saturated fatty acids and neuronal function.

Multiplex assays can provide beneficial information for basic cellular mechanisms&#160;and eliminate waste of reagents and unnecessary repetitive experiments. We describe here a multiplex caspase-3/7 activity assay, using fluorescent- and luminescent-based methods, to determine cell viability in an in vitro hypothalamic model following oxidative challenge with palmitic acid.

Utilização de uma caspase Multiplexing ensaio para determinar a apoptose em um modelo de célula hipotalâmica

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and ...

Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC regulates mitochondrial function and cellular energy status. Numerous reports demonstrated that the activation of PKC-a and PKC-&epsilon; improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-&alpha; and PKC-&epsilon; are involved in nephrotoxicant-induced mitochondrial dysfunction and cell death in kidney cells. Therefore, the goal of this study was to develop an in vitro model of renal cells maintaining active mitochondrial functions in which PKC isozymes could be selectively activated or inhibited to determine their role in regulation of oxidative phosphorylation and cell survival. Primary cultures of renal proximal tubular cells (RPTC) were cultured in improved conditions resulting in mitochondrial respiration and activity of mitochondrial enzymes similar to those in RPTC in vivo. Because traditional transfection techniques (Lipofectamine, electroporation) are inefficient in primary cultures and have adverse effects on mitochondrial function, PKC-&epsilon; mutant cDNAs were delivered to RPTC through adenoviral vectors. This approach results in transfection of over 90% cultured RPTC.
Here, we present methods for assessing the role of PKC-&epsilon; in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-&epsilon; is activated by overexpressing the constitutively active PKC-&epsilon; mutant. PKC-&epsilon; is inhibited by overexpressing the inactive mutant of PKC-&epsilon;. Mitochondrial function is assessed by examining respiration, integrity of the respiratory chain, activities of respiratory complexes and F0F1-ATPase, ATP production rate, and ATP content. Respiration is assessed in digitonin-permeabilized RPTC as state 3 (maximum respiration in the presence of excess substrates and ADP) and uncoupled respirations. Integrity of the respiratory chain is assessed by measuring activities of all four complexes of the respiratory chain in isolated mitochondria. Capacity of oxidative phosphorylation is evaluated by measuring the mitochondrial membrane potential, ATP production rate, and activity of F0F1-ATPase. Energy status of RPTC is assessed by determining the intracellular ATP content. Mitochondrial morphology in live cells is visualized using MitoTracker Red 580, a fluorescent dye that specifically accumulates in mitochondria, and live monolayers are examined under a fluorescent microscope. RPTC viability is assessed using annexin V/propidium iodide staining followed by flow cytometry to determine apoptosis and oncosis.
These methods allow for a selective activation/inhibition of individual PKC isozymes to assess their role in cellular functions in a variety of physiological and pathological conditions that can be reproduced in in vitro.

The effects of activation of protein kinase C (PKC) isozymes on mitochondrial functions associated with respiration and oxidative phosphorylation and on cell viability are described. The approach adapts adenoviral technique to selectively overexpress PKC isozymes in primary cell culture and a variety of assays to determine mitochondrial functions and energy status of the cell.

Avaliação de funções mitocondriais e viabilidade celular em células renais Proteína Kinase C superexpressão Isozimas

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC ...

Biologia

Viability Assays for Cells in Culture

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized viability assays are expensive in terms of equipment but can be faster and more objective than manual cell counts. The present report describes the use of three such viability assays. Two of these assays are infrared and one is luminescent. Both infrared assays rely on a 16&#160;bit Odyssey Imager. One infrared assay uses the DRAQ5 stain for nuclei combined with the Sapphire stain for cytosol and is visualized in the 700 nm channel. The other infrared assay, an In-Cell Western, uses antibodies against cytoskeletal proteins (&#945;-tubulin or microtubule associated protein 2) and labels them in the 800 nm channel. The third viability assay is a commonly used luminescent assay for ATP, but we use a quarter of the recommended volume to save on cost. These measurements are all linear and correlate with the number of cells plated, but vary in sensitivity. All three assays circumvent time-consuming microscopy and sample the entire well, thereby reducing sampling error. Finally, all of the assays can easily be completed within one day of the end of the experiment, allowing greater numbers of experiments to be performed within short timeframes. However, they all rely on the assumption that cell numbers remain in proportion to signal strength after treatments, an assumption that is sometimes not met, especially for cellular ATP. Furthermore, if cells increase or decrease in size after treatment, this might affect signal strength without affecting cell number. We conclude that all viability assays, including manual counts, suffer from a number of caveats, but that computerized viability assays are well worth the initial investment. Using all three assays together yields a comprehensive view of cellular structure and function.

Therapeutic compounds are often first examined in vitro with viability assays. Blind cell counts by a human observer can be highly sensitive to small changes in cell number but do not assess function. Computerized viability assays, as described here, can assess both structure and function in an objective manner.

Ensaios de viabilidade para células em cultura

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized ...

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 ...

An Introduction to Cell Death

Necrosis, apoptosis, and autophagic cell death are all manners in which cells can die, and these mechanisms can be induced by different stimuli, such as cell injury, low nutrient levels, or signaling proteins. Whereas necrosis is considered to be an &#8220;accidental&#8221; or unexpected form of cell death, evidence exists that apoptosis and autophagy are both programmed and &#8220;planned&#8221; by cells.In this introductory video, JoVE highlights key discoveries pertaining to cell death, including recent work done in worms that helped identify genes involved in apoptosis. We then explore questions asked by scientists studying cell death, some of which look at different death pathways and their interactions. Finally, several methods to assess cell death are discussed, and we note how researchers are applying these techniques in their experiments today.

Cell Death: Apoptosis, Autophagy and Necrosis

Uma introdução à morte celular

Necrosis, apoptosis, and autophagic cell death are all manners in which cells can die, and these mechanisms can be induced by different stimuli, such as ...

Education

JoVE Science Education

Biologia Avançada

Biologia Celular

Annexin V and Propidium Iodide Labeling

Staining with annexin V and propidium iodide (PI) provides researchers with a way to identify different types of cell death&#8212;either necrosis or apoptosis. This technique relies on two components. The first, annexin V, is a protein that binds certain phospholipids called phosphatidylserines, which normally occur only in the inner, cytoplasm-facing leaflet of a cell&#8217;s membrane, but become &#8220;flipped&#8221; to the outer leaflet during the early stages of apoptosis. The second component is the DNA-binding dye molecule PI, which can only enter cells when their membranes are ruptured&#8212;a characteristic of both necrosis and late apoptosis.This video article begins with a review of the concepts behind annexin V and PI staining, and emphasizes how differential patterns of staining can be used to distinguish between cells progressing down different death pathways. We then review a generalized protocol for this technique, followed by a description of how researchers are currently using annexin V and PI staining to better understand cell death.

Cell Death Pathways and Annexin V &amp; PI Labeling studies

Marcação com Anexina V e Iodeto de Propídio

Staining with annexin V and propidium iodide (PI) provides researchers with a way to identify different types of cell death&#8212;either necrosis or ...