Name: immunofluorescence microscopy of γh2ax and 53bp1 for analyzing the formation and repair of dna double-strand breaks
Uploaded: 2017-11-03T14:00:00.000+00:00
Duration: 10 min 47 s
Description: Watch this Scientific Journal Video about Immunofluorescence Microscopy of Î³H2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks at JoVE.com

O projeto foi apoiado pela Fundação de leucemia do alemão José Carreras (14 DJCLS R/2017).

todos os métodos descritos aqui foram aprovados pelo Comitê ética II de a Mannheim faculdade médica da Universidade de Heidelberg. Escrito foi obtido o consentimento de todos os indivíduos.
 1. preparação de materiais <ol><li>solução anticoagulante: preparar uma solução anticoagulante de 200 heparina UI / mL de cloreto de sódio 0,9%. Encha cada um dos tubos de coleta (desenhar volume 9 mL) com 2 mL da solução anticoagulante antes da retirada das amostras de sangue ou de medula óssea.</li>
	<li>Solução de lise de eritrócitos: preparar a 10 x solução de lise de eritrócitos com 82,91 g de cloreto de amônio, bicarbonato de amónio 7,91 g e 2 mL de solução de ácido etilenodiaminotetracético 0,5 M para um pH 8.0, dissolvendo os agentes em água bidestilada, para um volume total de 1 L.</li>
	<li>Solução de fixação: adicionar 8,3 µ l de uma solução de hidróxido de potássio 1m de paraformaldeído 360 mg (PFA) em um tubo de microtitulação. Encha o tubo com solução salina tamponada fosfato (PBS) até a marca de 1 mL do tubo e calor do tubo em um bloco de aquecimento a 95 ° C para 1 mL de uma solução PFA de 36%. Transferi a solução PFA 36% 1 mL para um tubo com uma capacidade de 15 mL. Adicione 8 mL de PBS para a solução PFA de 36% de 1 mL, para obter 9 mL de uma solução stock de PFA 4%. Alíquota da solução estoque de PFA de 4% e loja a-18 ° C até usam para a fixação das células. 
	Cuidado: solução 1) hidróxido de potássio é corrosiva. Esteja ciente de proteção da pele e olhos. 2) PFA é cancerígeno. Evite a inalação e contacto com a pele. Preparar a solução de fixação sob um capô.</li>
	<li>Solução de permeabilização: adicionar 50 µ l Octoxinol 9 a 50 mL PBS para obter uma solução de aproximadamente 0,1% Octoxinol 9.</li>
	<li>Obstrui a solução: preparar 5% e 2% de bloqueio soluções misturando o agente bloqueio de proteína com PBS e loja em 6 &#8211; 8 ° C.</li>
</ol> 2. Preparação das amostras <ol><li>fibroblastos em cultura de células: cultivar fibroblastos humanos da linha celular NHDF em um umidificado 5% CO 2 atmosfera a 37 ° C em meio RPMI 1640, suplementado com 10% de soro fetal bezerro, 4 mM glutamina e 1% penicilina/estreptomicina.
	<ol><li>a preparação de uma lâmina de microscópio com aderente fibroblastos <ol><li>remover o meio do balão (área de crescimento de 2 de 75 cm), com crescimento exponencial fibroblastos NHDF. Adicione 10 mL de PBS no balão. Lave os fibroblastos aderentes suavemente agitando manualmente o frasco de 1 min.</li>
			<li>Remover a PBS e adicionar tripsina 1 mL para o frasco. Agite o frasco com cuidado para assegurar que todas as células aderentes são cobertas por tripsina. Retire o balão a tripsina. Colocar o balão em incubadora para 5 min a 37 ° C.</li>
			<li>Tomar o frasco fora da incubadora e adicionar 10 mL de RPMI 1640 de meio para o balão. Pipetar o meio subindo e descendo várias vezes para dispersar os fibroblastos.</li>
			<li>Pipetar 0,3 mL dos fibroblastos dispersos em cada um dos dois campos da lâmina de microscópio e colocar o slide em incubadora para 24h para que os fibroblastos aderirem ao slide. Para imunocoloração de γH2AX e 53BP1 em núcleos de fibroblastos, avançar para o passo 3.1.</li>
		</ol></li></ol></li> <li>Amostras de pacientes <ol><li>amostras de sangue: usar os tubos de coleta que foram preenchidos com 2 mL da solução anticoagulante e adicionar sangue 7 mL para os tubos. Armazene as amostras durante a noite em temperatura ambiente (ou seja, 18-20 ° C). No dia seguinte, isolar a fase G0/G1 descansando células mononucleares por centrifugação gradiente de densidade (etapa 2.2.3).</li>
		<li>Amostras de medula óssea: usar os tubos de coleta que foram preenchidos com 2 mL da solução anticoagulante (etapa 1.1) e adicionar 7 mL medula para os tubos. Armazene as amostras durante a noite em temperatura ambiente (ou seja, 18-20 ° C). No dia seguinte, isole a fase G0/G1 descansando células mononucleares por centrifugação gradiente de densidade (etapa 2.2.3). Use microbeads CD34 e colunas de separação para o isolamento de células CD34 + da pilha.</li>
		<li>Isolamento de células mononucleares por centrifugação gradiente de densidade 
		Nota: As células mononucleares são isoladas de amostras de sangue e medula óssea por centrifugação gradiente de densidade 17 , 18 , 19.
		<ol><li>Diluir a amostra de sangue heparinizado em uma proporção de 1:1 com PBS e a amostra heparinizada medula óssea na proporção de 1:3 com PBS. Suspender as células suavemente desenhando-as dentro e fora da pipeta duas vezes.</li>
			<li>Adicionar um volume de meio de gradiente de densidade para um tubo de fresco. Camada delicadamente o sangue diluído ou medula óssea em cima do meio de gradiente de densidade. Tome cuidado para não misturar as duas camadas.</li>
			<li>Centrifugar o tubo durante 30 min à temperatura ambiente e 400 g. x desviar a camada superior de plasma com a pipeta. Então, cuidadosamente Colha as células mononucleares na camada acima do meio de gradiente de densidade. Transferir as células mononucleares em um tubo fresco.</li>
			<li>Adicionar pelo menos três volumes de PBS para as células mononucleares no tubo. Suspenda as células suavemente desenhando-as dentro e fora da pipeta duas vezes. Centrifugar o tubo para 10 min a temperatura ambiente e 400 g. x</li>
			<li>Após o spin remover o sobrenadante e adicionar 10 mL de 4 ° C, 1 x solução de lise de eritrócitos. Ressuspender as células suavemente desenhando-as dentro e fora da pipeta duas vezes e colocar o tubo no gelo por 5 min. Em seguida, adicionar 30 mL de PBS a 4 ° C e centrifugar o tubo para 10 min a temperatura ambiente e 400 g. x</li>
		</ol></li> <li>Preparação da cytospins <ol><li>preparar dois cytospins com células mononucleares das amostras (ou seja, um cytospin para coloração de imunofluorescência de γH2AX e um cytospin para γH2AX e 53BP1 combinado a mancha da imunofluorescência) por centrifugação (300 x g, 10 min) de 1,0 x 10 5 células para cada preparação ( Figura 3 e 4).</li>
		</ol></li></ol></li></ol> 3. γH2AX e coloração de imunofluorescência de 53BP1 <ol><li>fixação e permeabilização: consertar as células com 200 µ l de 4% PFA por 10 min. Após a fixação, lave suavemente as células três vezes com 30 mL de PBS por 5 min num agitador de laboratório. Em seguida, permeabilize as células com 200 µ l de 0.1% Octoxinol 9 de 10 min. lavar as células suavemente três vezes com 30 mL de solução de bloqueio de 5% por 5 min num agitador de laboratório. Bloquear as células em 30 mL de fresco 5%, que obstrui a solução por 1 h.</li>
	<li>De incubação com anticorpos <ol><li>incuba uma preparação das células com um anticorpo monoclonal anti-γH2AX de rato (1: 500) e a outra preparação das células com um anticorpo monoclonal anti-γH2AX de rato (1: 500) e um anticorpo de coelho policlonal anti-53BP1 (1: 500) durante a noite em 4 ° C.</li>
		<li>Após incubação lavar as células suavemente 3 vezes com 30 mL de solução de bloqueio de 2% por cada 5 min em um shaker laboratório.</li>
		<li>Remover a solução de bloqueio e incubar a primeira preparação das células com um anticorpo secundário de anti-rato conjugada Alexa488 cabra (1: 500) e a segunda preparação das células com uma cabra Alexa488-conjugado anticorpo secundário anti-rato ( 1: 500) e um burro Alexa555-conjugado anticoelho anticorpo secundário (1: 500) por 1h à temperatura ambiente.</li>
	</ol></li> <li>Meio de montagem: Coloque o slide com as células em uma tigela e lave as células suavemente três vezes com 30 mL de PBS para cada 5 min num agitador de laboratório. Remover a PBS e montar as células com o meio de montagem contendo 4,6-diamidino-2-phenylindole. Cautelosamente, colocar uma lamela em cima do meio de montagem para que nenhuma bolha de ar incorporado. Espere pelo menos 3 h para endurecimento do meio de montagem antes de analisar as células por microscopia de fluorescência.</li>
</ol> 4. Análise de γH2AX e 53BP1 focos <ol><li>microscopia de fluorescência: analisar os focos de γH2AX e 53BP1 no núcleo de células com um microscópio de fluorescência, equipado com filtros para DAPI, Alexa 488 e Cy3 durante a imagem latente em um objetivo X 100 ampliação. Gravar imagens com uma câmera e processar as imagens com o software de imagem adequado.</li>
</ol>

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Microscopia de imunofluorescência de γH2AX e 53BP1 é um método útil para analisar a formação e reparação de ORL em um amplo espectro de domínios de investigação. Parâmetros críticos que influenciam o resultado dos experimentos são a fase do ciclo celular, os agentes utilizados para a fixação e permeabilização das células, a escolha dos anticorpos e o hardware e software, o microscópio de fluorescência.
A influência do ciclo celular em padrões de focos γH2AX foi demonst...

O DNA é danificado continuamente por endógenos (por exemplo, estresse de replicação, espécies reativas de oxigênio, instabilidade intrínseca do DNA) e exógenos (por exemplo, os radicais químicos, irradiação) fontes (Figura 1)1,2 ,3,4. Entre os danos do DNA, DNA dupla quebras (DSB) são particularmente graves lesões e podem induzir a morte celular ou carcinogênese. Cerca de 50 DSB pode surgir por célula e ciclo celular5. Em células de mamíferos, recombinação homóloga (HR) e não-homólogos final-adesão (NHEJ) desenvolveram-se como principais vias para a reparação de ORL (Figura 2). RH ocorre no final da fase S/G2 e usa uma irmã intacta cromátides irmãs como um modelo para reparação potencialmente livre de erros. Em comparação, NHEJ é ativo ao longo do ciclo celular e potencialmente mutagénicas como pares de base pode ser adicionados ou ressecados antes da ligadura das extremidades quebradas. Além disso, final alternativo-adesão pode ser contratado como um mecanismo lento mutagénicas backup reparação em caso de deficiência NHEJ6,7.
DSB induzir a fosforilação da histona H2AX em serina 139 em uma região de vários pares de megabase ao redor de cada DSB. Os focos de formação nucleares são denominados focos de γH2AX e são detectáveis por microscopia de imunofluorescência8. ΓH2AX promove o recrutamento de mais proteínas DSB-responsiva e está envolvido na cromatina remodelação, reparação de DNA e de transdução de sinal9. Como cada foco de γH2AX é considerado para representar um único DSB, a quantificação do DSB por microscopia de imunofluorescência é possível, que tem sido demonstrada em linhas de células de câncer e espécimes paciente10,11, 12 , 13 , 14. 53BP1 (p53 binding protein 1) é outra proteína-chave na mediação reparação de ORL. Está envolvido no recrutamento de DSB-responsivo proteínas, sinalização de ponto de verificação e a sinapse de ORL termina15. Além disso, 53BP1 desempenha um papel crítico na escolha de caminho de reparação de ORL. Ele aciona a reparação de ORL para NHEJ enquanto HR é impedido de16. Considerando as funções genuínas de γH2AX e 53BP1 no reparo DSB, a análise simultânea de γH2AX e 53BP1 por microscopia de imunofluorescência pode ser um método útil para a análise precisa da formação e reparação de ORL.
Este manuscrito fornece um protocolo passo a passo para a realização de microscopia de imunofluorescência de γH2AX e 53BP1 no núcleo de células. Especificamente, a técnica é aplicada em fibroblastos normais da linha celular NHDF, em linfócitos de raio-x irradiado de um indivíduo saudável e em células CD34 + de um paciente com leucemia mieloide aguda. Os detalhes do método são apontados no contexto dos resultados apresentados.

<table><tbody><tr><td>RPMI medium</td><td>Sigma-Aldrich</td><td>R0883</td><td>Medium for cell culture</td></tr><tr><td>Heparin sodium</td><td>ratiopharm</td><td>PZN 3029843</td><td>&amp;nbsp;Heparin 5,000 I.U. / mL</td></tr><tr><td>Sodium chloride solution 0.9%</td><td>B. Braun</td><td>PZN 1957154</td><td>Component of the anticoagulant stock solution</td></tr><tr><td>Ficoll-Paque Premium</td><td>GE Healthcare</td><td>17-5442-02</td><td>Medium for isolation of mononuclear cells</td></tr><tr><td>Trypsin solution 10X</td><td>Sigma-Aldrich</td><td>59427C</td><td>Enzyme for dissociation of fibroblasts in cell culture</td></tr><tr><td>CD34 MicroBead Kit</td><td>Miltenyi Biotec</td><td>130-046-702</td><td>Isolation of CD34+ myeloid progenitor cells</td></tr><tr><td>Diagnostic microscope slides</td><td>Thermo Scientific</td><td>ER-203B-CE24</td><td>Microscope slides</td></tr><tr><td>Megafuge 1.0 R</td><td>Heraeus</td><td>75003060</td><td>&amp;nbsp;Tabletop centrifuge&amp;nbsp;</td></tr><tr><td>Cytospin device</td><td></td><td></td><td></td></tr><tr><td>Lid</td><td>Heraeus</td><td>76003422</td><td>Lid for working without micro-tubes</td></tr><tr><td>Cyto container</td><td>Heraeus</td><td>75003416</td><td>Cyto container with 2 conical bores</td></tr><tr><td>Clip carrier</td><td>Heraeus</td><td>75003414</td><td>Carrier for holding a cyto container and a slide</td></tr><tr><td>Support insert</td><td>Heraeus</td><td>75003417</td><td>Support insert for holding a clip carrier</td></tr><tr><td>Triton X-100 (Octoxinol 9)</td><td>Thermo Scientific</td><td>85112</td><td>Detergent for permeabilization of cell membranes</td></tr><tr><td>Potassium hydroxide solution 1M</td><td>Merck Millipore</td><td>109107</td><td>Necessary for preparing the paraformaldehyde solution</td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td>Fixation agent</td></tr><tr><td>Phosphate buffered saline</td><td>Sigma-Aldrich</td><td>D8537</td><td>Balanced salt solution</td></tr><tr><td>Chemiblocker</td><td>Merck Millipore</td><td>2170</td><td>Blocking agent</td></tr><tr><td>Mouse monoclonal anti-&amp;gamma;H2AX antibody (JBW301)</td><td>Merck Millipore</td><td>05-636</td><td>Primary antibody for detection of &amp;gamma;H2AX</td></tr><tr><td>Polyclonal rabbit anti-53BP1 antibody (NB100-304)</td><td>Novus Biologicals</td><td>NB100-304</td><td>Primary antibody for detection of 53BP1</td></tr><tr><td>Alexa Fluor 488-conjugated goat anti-mouse antibody</td><td>Invitrogen</td><td>A-11001</td><td>Secondary antibody</td></tr><tr><td>Alexa Fluor 555-conjugated donkey anti-rabbit antibody</td><td>Invitrogen</td><td>A-31572</td><td>Secondary antibody</td></tr><tr><td>Vectashield mounting medium</td><td>Vector Laboratories</td><td>H-1200</td><td>Contains DAPI for staining of DNA</td></tr><tr><td>Axio Scope.A1</td><td>Zeiss</td><td>490035</td><td>Fluorescence microscope</td></tr><tr><td>Cool Cube 1 CCD camera</td><td>Metasystems</td><td>H-0310-010-MS</td><td>Camera system for digital recording</td></tr><tr><td>Isis software</td><td>Metasystems</td><td>Not applicable</td><td>Microscope software</td></tr></tbody></table>

immunofluorescence microscopy of &#947;h2ax and 53bp1 for analyzing the formation and repair of dna double-strand breaks

Quebras de DNA dobro-Costa (DSB) são graves lesões do ADN. Análise da formação e reparação do DSB é relevante em um amplo espectro de domínios de investigação, incluindo a integridade do genoma, genotoxicidade, biologia da radiação, envelhecimento, câncer e desenvolvimento de drogas. Em resposta ao ORL, o histona H2AX é fosforilado em serina 139 em uma região de vários pares de megabase formando discretos focos nucleares detectáveis pela microscopia de imunofluorescência. Além disso, 53BP1 (p53 binding protein 1) é outra importante proteína DSB-responsivo promover a reparação de DSB, juntando-se não-homólogos final-evitando a recombinação homóloga. De acordo com as funções específicas de γH2AX e 53BP1, a análise combinada de γH2AX e 53BP1 por microscopia de imunofluorescência pode ser uma abordagem razoável para uma análise detalhada de ORL. Este manuscrito fornece um protocolo passo a passo, complementado com notas metódicas para executar a técnica. Especificamente, a influência do ciclo celular em padrões de focos γH2AX é demonstrada em fibroblastos normais da linha celular NHDF. Além disso, o valor dos focos de γH2AX como um biomarcador é retratado em linfócitos de raio-x irradiado de um indivíduo saudável. Finalmente, a instabilidade genética é investigada em células CD34 + de um paciente com leucemia mieloide aguda por microscopia de imunofluorescência de γH2AX e 53BP1.

Análise dos focos de γH2AX nas células é mais preciso na fase G0/G1 e a G2 fase quando γH2AX focos aparecem como distintos pontos fluorescentes (Figura 5A). Em contraste, análise dos focos de γH2AX nas células durante a fase S é complicado por manchas dispersas γH2AX pan-nuclear causadas pelo processo de replicação (Figura 5B).
Fixação das células foi rea...

Watch this Scientific Journal Video about Immunofluorescence Microscopy of Î³H2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks at JoVE.com

Immunofluorescence Microscopy of &#947;H2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks

Este manuscrito fornece um protocolo de análise de quebras de DNA dobro-costa pela microscopia de imunofluorescência de γH2AX e 53BP1.

Microscopia de imunofluorescência de γH2AX e 53BP1 para analisar a formação e reparação do DNA Double-strand Breaks

DNA double-strand breaks (DSB) are serious DNA lesions. Analysis of the formation and repair of DSB is relevant in a broad spectrum of research areas ...

immunofluorescence-microscopy-h2ax-53bp1-for-analyzing-formation

Universidad San Sebastian

Research

JoVE Journal

Cancer Research

16.1K visualizações.  Medical Faculty Mannheim of the Heidelberg University. Este manuscrito fornece um protocolo de análise de quebras de DNA dobro-costa pela microscopia de imunofluorescência de γH2AX e 53BP1.

Vídeo: Immunofluorescence Microscopy of γH2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks

Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair complexes. This process is regulated by a myriad of proteins that promote the association and disassociation of proteins to these lesions. Thanks in large part to the ability to perform functional screens of a vast library of proteins, there is a greater appreciation of the genes necessary for the double-strand DNA break repair. Often knockout or chemical inhibitor screens identify proteins involved in repair processes by using increased toxicity as a marker for a protein that is required for DSB repair. Although useful for identifying novel cellular proteins involved in maintaining genome fidelity, functional analysis requires the determination of whether the protein of interest promotes localization, formation, or resolution of repair complexes. 
The accumulation of repair proteins can be readily detected as distinct nuclear foci by immunofluorescence microscopy. Thus, association and disassociation of these proteins at sites of DNA damage can be accessed by observing these nuclear foci at representative intervals after the induction of double-strand DNA breaks. This approach can also identify mis-localized repair factor proteins, if repair defects do not simultaneously occur with incomplete delays in repair. In this scenario, long-lasting double-strand DNA breaks can be engineered by expressing a rare cutting endonuclease (e.g., I-SceI) in cells where the recognition site for the said enzyme has been integrated into the cellular genome. The resulting lesion is particularly hard to resolve as faithful repair will reintroduce the enzyme's recognition site, prompting another round of cleavage. As a result, differences in the kinetics of repair are eliminated. If repair complexes are not formed, localization has been impeded. This protocol describes the methodology necessary to identify changes in repair kinetics as well as repair protein localization.

Repair of double-strand DNA breaks is a dynamic process, requiring not only formation of repair complexes at the breaks, but also their resolution after the lesion is addressed. Here, we use immunofluorescence microscopy for transient and long-lasting double-stranded breaks as a tool to dissect this genome maintenance mechanism.

Caracterizando os processos de reparação do DNA no transitório e duradouro dobro-Costa quebras de DNA pela microscopia de imunofluorescência

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair ...

Pesquisa do câncer

Immunofluorescence Imaging of DNA Damage and Repair Foci in Human Colon Cancer Cells

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage response induced by neutron-gamma mixed-beam used in boron neutron capture therapy (BNCT). Specifically, the proposed methodology is applied for the detection of repair proteins activation which can be visualized as foci using antibodies specific to DNA double-strand breaks (DNA-DSBs). DNA repair foci were assessed by immunofluorescence in colon cancer cells (HCT-116) after irradiation with the neutron-mixed beam. DNA-DSBs are the most genotoxic lesions and are repaired in mammalian cells by two major pathways: non-homologous end-joining pathway (NHEJ) and homologous recombination repair (HRR). The frequencies of foci, immunochemically stained, for commonly used markers in radiobiology like &#947;-H2AX, 53BP1 are associated with DNA-DSB number and are considered as efficient and sensitive markers for monitoring the induction and repair of DNA-DSBs. It was established that &#947;-H2AX foci attract repair proteins, leading to a higher concentration of repair factors near a DSB. To monitor DNA damage at the cellular level, immunofluorescence analysis for the presence of DNA-PKcs representative repair protein foci from the NHEJ pathway and Rad52 from the HRR pathway was planned. We have developed and introduced a reliable immunofluorescence staining protocol for the detection of radiation-induced DNA damage response with antibodies specific for repair factors from NHEJ and HRR pathways and observed radiation-induced foci (RIF). The proposed methodology can be used for investigating repair protein that is highly activated in the case of neutron-mixed beam radiation, thereby indicating the dominance of the repair pathway.

Radiation-induced DNA damage response activated by neutron mixed-beam used in boron neutron capture therapy (BNCT) has not been fully established. This protocol provides a step-by-step procedure to detect radiation-induced foci (RIF) of repair proteins by immunofluorescence staining in human colon cancer cell lines after irradiation with the neutron-mixed beam.

Imagens de imunofluorescência de dna dano e reparo de focos em células cancerígenas de cólon humano

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage ...

An Automated Microscopic Scoring Method for the &#947;-H2AX Foci Assay in Human Peripheral Blood Lymphocytes

Ionizing radiation is a potent inducer of DNA damage and a well-documented carcinogen. Biological dosimetry comprises the detection of biological effects induced by exposure to ionizing radiation to make an individual dose assessment. This is pertinent in the framework of radiation emergencies, where health assessments and planning of clinical treatment for exposed victims are critical. Since DNA double strand breaks (DSB) are considered to be the most lethal form of radiation-induced DNA damage, this protocol presents a method to detect DNA DSB in blood samples. The methodology is based on the detection of a fluorescent labelled phosphorylated DNA repair protein, namely, &#947;-H2AX. The use of an automated microscopy platform to score the number of &#947;-H2AX foci per cell allows a standardized analysis with a significant decrease in the turn-around time. Therefore, the &#947;-H2AX assay has the potential to be one of the fastest and sensitive assays for biological dosimetry. In this protocol, whole blood samples from healthy adult volunteers will be processed and irradiated in vitro in order to illustrate the usage of the automated and sensitive &#947;-H2AX foci assay for biodosimetry applications. An automated slide scanning system and analysis platform with an integrated fluorescence microscope is used, which allows the fast, automatic scoring of DNA DSB with a reduced degree of bias.

This protocol presents the slide preparation and automated scoring of the &#947;-H2AX foci assay on peripheral blood lymphocytes. To illustrate the method and sensitivity of the assay, isolated lymphocytes were irradiated in vitro. This automated method of DNA DSB detection is useful for fast and high-throughput biological dosimetry applications.

Um método automatizado de pontuação microscópica para o ensaio de γ-H2AX Foci em Linfócitos de Sangue Periféricos Humanos

Ionizing radiation is a potent inducer of DNA damage and a well-documented carcinogen. Biological dosimetry comprises the detection of biological effects ...

Dual Immunofluorescence of &#947;H2AX and 53BP1 in Human Peripheral Lymphocytes

Double strand breaks (DSBs) are one of the most severe lesions that can occur in cell nuclei, and, if not repaired, they can lead to severe outcomes, including cancer. The cell is, therefore, provided with complex mechanisms to repair DSBs, and these pathways involve histone H2AX in its phosphorylated form at Ser-139 (namely &#947;H2AX) and p53 binding protein 1 (53BP1). As both proteins can form foci at the sites of DSBs, identification of these markers is considered a suitable method to study both DSBs and their kinetics of repair. According to the molecular processes that lead to the formation of &#947;H2AX and 53BP1 foci, it could be more useful to investigate their co-localization near the DSBs in order to set up an alternative approach that allows quantifying DSBs by the simultaneous detection of two DNA damage markers. Thus, this protocol aims to assess the genomic damage induced in human lymphocytes by the radiomimetic agent bleomycin through the presence of &#947;H2AX and 53BP1 foci in a dual immunofluorescence. Using this methodology, we also delineated the variation in the number of &#947;H2AX and 53BP1 foci over time, as a preliminary attempt to study the repair kinetics of bleomycin-induced DSBs.

This protocol presents a method to assess the formation and repair of DNA double-strand breaks through the simultaneous detection of &#947;H2AX and 53BP1 foci in interphase nuclei of bleomycin-treated human peripheral lymphocytes.

Imunofluorescência Dupla de γH2AX e 53BP1 em Linfócitos Periféricos Humanos

Double strand breaks (DSBs) are one of the most severe lesions that can occur in cell nuclei, and, if not repaired, they can lead to severe outcomes, ...

Genética

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

Quantificação de γH2AX Focos em resposta à radiação ionizante

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Biologia

Quantitation of &gamma;H2AX Foci in Tissue Samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX, is an early response to DNA double-strand breaks1. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Overall, DSB induction results in the formation of discrete nuclear &gamma;H2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2. Given the unique specificity and sensitivity of this marker, analysis of &gamma;H2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of &gamma;H2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, &gamma;H2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of &gamma;H2AX foci ex vivo and in vivo4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of &gamma;H2AX foci in mouse tissues.

Quantitation of &#947;H2AX Foci in Tissue Samples

Quantitation of DNA double-strand breaks on the basis of &gamma;H2AX foci has become an invaluable tool, particularly in radiation biology, for the evaluation of tissue radiosensitivity and effects of radiation modifying compounds. Here we demonstrate the use of an immunofluorescence assay for quantitation of &gamma;H2AX foci in tissue samples.

Quantificação de γH2AX Focos em amostras de tecido

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) ...

Evaluation of the Spatial Distribution of &gamma;H2AX following Ionizing Radiation

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3. The phosphorylated form of H2AX, referred to as &gamma;H2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3. Analysis and quantitation of &gamma;H2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds4.
Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of &gamma;H2AX as molecular marker of DSBs, a disparity in &gamma;-irradiation-induced &gamma;H2AX foci formation in euchromatin and heterochromatin has been observed5-7. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of &gamma;H2AX following ionizing radiation8. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between &gamma;H2AX formation and active transcription9. Here we demonstrate our immunofluorescence method for detection and quantitation of &gamma;H2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3D-modeling.

Evaluation of the Spatial Distribution of &#947;H2AX following Ionizing Radiation

Microscopic analysis of &gamma;H2AX foci, which form following the phosphorylation of H2AX at Ser-139 in response to DNA double-strand breaks, has become an invaluable tool in radiation biology. Here we used an antibody to mono-methylated histone H3 at lysine 4 as an epigenetic marker of actively transcribing euchromatin, to evaluate the spatial distribution of radiation-induced &gamma;H2AX formation within the nucleus.

Avaliação da Distribuição Espacial da γH2AX seguintes Radiação Ionizante

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone ...

Visualization of DNA Repair Proteins Interaction by Immunofluorescence

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA insults. Genotoxic agents can damage the DNA backbone, break it, or modify the chemical nature of individual bases. Following DNA insult, DNA damage response (DDR) pathways are activated and proteins involved in the repair are recruited. A plethora of factors are involved in sensing the type of damage and activating the appropriate repair response. Failure to correctly activate and recruit DDR factors can lead to genomic instability, which underlies many human pathologies including cancer. Studies of DDR proteins can provide insights into genotoxic drug response and cellular mechanisms of drug resistance.
There are two major ways of visualizing&#160;proteins in vivo: direct observation, by tagging the protein of interest with a fluorescent protein and following it by live imaging, or indirect immunofluorescence on fixed samples. While visualization of fluorescently tagged proteins allows precise monitoring over time, direct tagging in N- or C-terminus can interfere with the protein localization or function. Observation of proteins in their unmodified, endogenous version is preferred. When DNA repair proteins are recruited to the DNA insult, their concentration increases locally and they form groups, or &#8220;foci&#8221;, that can be visualized by indirect immunofluorescence using specific antibodies.
Although detection of protein foci does not provide a definitive proof of direct interaction, co-localization of proteins in cells indicates that they regroup to the site of damage and can inform of the sequence of events required for complex formation. Careful analysis of foci spatial overlap in cells expressing wild type or mutant versions of a protein can provide precious clues on functional domains important for DNA repair function. Last, co-localization of proteins indicates possible direct interactions that can be verified by co-immunoprecipitation in cells, or direct pulldown using purified proteins.

Following DNA damage, human cells activate essential repair pathways to restore the integrity of their genome. Here, we describe the method of indirect immunofluorescence as a means to detect DNA repair proteins, analyze their spatial and temporal recruitment, and help interrogate protein-protein interaction at the sites of DNA damage.

Visualização de Proteínas de Reparação de DNA Interação por Imunofluorescência

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA ...

<meta charset="utf8"></head><body>Avaliando a interação e localização de proteínas em danos ao DNA

Proximity Ligand Assay to Localize Proteins in DNA Damage Sites

The DNA damage response is a genetic information safeguard that protects cells from perpetuating damaged DNA. The characterization of the proteins that cooperate in this process allows the identification of alternative targets for therapeutic intervention in several diseases, such as cancer, aging-related diseases, and chronic inflammation. The Proximity Ligand Assay (PLA) emerged as a tool for estimating interaction between proteins as well as spatial proximity among organelles or cellular structures and allows the temporal localization and co-localization analysis under stress conditions, for instance. The method is simple because it is similar to conventional immunofluorescence and allows the staining of an organelle, cellular structure, or a specific marker such as mitochondria, endoplasmic reticulum, PML bodies, or DNA double-strand marker, yH2AX simultaneously. The phosphorylation of the S139 at Histone 2A variant, H2AX, then referred to as yH2AX, is widely used as a very sensitive and specific marker of DNA double-strand breaks. Each focus of yH2AX staining corresponds to one break in DNA that occurs a few minutes after the damage. The analysis of changes in yH2AX foci is the most common assay for studying if the protein of interest is implicated in DNA damage response (DDR). Whether a direct role in the DNA damage site is expected, fluorescence microscopy is used to verify the colocalization of the protein of interest with yH2AX foci. However, except for the new super-resolution fluorescence methods, to conclude, the local interaction with DNA damage sites can be a little subjective. Here, we show an assay to evaluate the localization of proteins in the DDR pathway using yH2AX as a marker of the damage site. This assay can be used to characterize the temporal localization under different insults that cause DNA damage.

<meta charset="utf8"></head><body>Autor em destaque: Combinando ensaio de ligante de proximidade com coloração gama-H2AX para caracterizar interações proteicas na resposta a danos no DNA

Here, we present a simple and rapid protocol for the detection of protein interaction at DNA damage sites.

Ensaio de ligante de proximidade para localizar proteínas em locais de dano ao DNA

The DNA damage response is a genetic information safeguard that protects cells from perpetuating damaged DNA. The characterization of the proteins that ...

Microscopia de imunofluorescência de γH2AX e 53BP1 para analisar a formação e reparação do DNA Double-strand Breaks

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