O apoio do Instituto Australiano de Ciência Nuclear e Engenharia é reconhecido. TCK foi o ganhador de prêmios AINSE. Lab Medicine epigenômico é suportado pelo National Health and Medical Research Council of Australia (566.559). LM é apoiada por Melbourne Research (University of Melbourne) e Biomedical Imaging bolsas complementares CRC. O apoio de Monash Micro Imaging (Drs. Stephen Cody e ISKA Carmichael) foi inestimável para este trabalho.

<html> Preparação de células <ol><li> Queratinócitos humanos (FEP-1811) foram cultivadas em queratinócitos-Serum Médio gratuito (K-SFM; Invitrogen) suplementado com fator de crescimento epidérmico, o extrato de hipófise de bovinos e 20 mcg / ml de gentamicina, a 37 ° CO C e 5% 2. </li><li> A suspensão única célula foi preparado por destacando com tripsina-EDTA (0,05% v / v) </li><li> Células foram semeadas em oito bem-Lab Tek slides II MicroChamber (10.000 células / poço) e as lâminas foram incubadas por 3 dias a 37 ° C e 5% de CO 2. </li></ol> Irradiação <ol><li> As células foram irradiadas no gelo com 2 Gy usando uma fonte de 137 Cs (Gammacell irradiador 1000 Elite; Nordion International, ON, Canadá, 20,6 segundos / Gy) </li><li> Controle não irradiado e 2 Gy células irradiadas foram incubadas por 1 hora a 37 ° C e 5% CO 2. </li></ol> Imunofluorescência <ol><li> Mídia foi derrubado fora e as células foram lavadas com 300 ul de PBS (w / o Ca 2 + ou Mg 2 +) por poço e foram rodados em um misturador orbital por 5 minutos. </li><li> O buffer foi derrubado fora e 100μl de recém-preparados 4% (v / v) paraformaldeído foi adicionado a cada poço e as lâminas foram incubadas em temperatura ambiente por 10 minutos. Todas as incubações foram realizadas em um cocho de coloração umidificado </li><li> As células foram então lavadas com PBS (w / o Ca 2 + ou Mg 2 +). Lâminas foram colocadas em um frasco Coplin e rodado em um misturador orbital por 5 minutos. Esta etapa de lavagem foi repetido mais duas vezes. </li><li> O buffer foi derrubado fora e excesso de PBS foi gentilmente apagados. </li><li> Células foram permeabilizadas com 100ml Triton X-100 (0,1% v / v) por poço e uma incubação de 10 minutos em temperatura ambiente. </li><li> Células foram lavadas com PBS (w / o Ca 2 + ou Mg 2 +), conforme descrito nos passos 3 e 4 acima. </li><li> Ligação não específica de proteínas foi bloqueada com 100 ml de BSA (1% v / v) por poço e 20 minutos de incubação à temperatura ambiente. </li><li> BSA excesso foi derrubado fora e 100μl do ensino primário monoclonal anti-anticorpo fosfo-histona H2AX (diluído 1:500 em BSA 1%; Millipore), foi adicionado a cada poço para a incubação de uma hora à temperatura ambiente. </li><li> Células foram lavadas com PBS (w / o Ca 2 + ou Mg 2 +) conforme descrito nas etapas 3 e 4 acima e incubadas com 100μl de anticorpo secundário (Alexa Fluor 488 cabra anti-camundongo IgG diluído 1:500 em BSA 1% ; Invitrogen) por poço por 45 minutos em temperatura ambiente no escuro. (Anticorpo diluído foi mantido no escuro durante todo o procedimento) </li><li> Células foram lavadas com PBS (w / o Ca 2 + ou Mg 2 +), conforme descrito nos passos 3 e 4 acima. No entanto, a exposição à luz foi minimizado usando papel alumínio. </li><li> Contracoloração nuclear foi realizado com 100 ml TOPRO3 (diluído 1:500; Invitrogen) por poço e incubação de 10 minutos em temperatura ambiente </li><li> Células foram lavadas com PBS (w / o Ca 2 + ou Mg 2 +) como descrito na etapa 10 acima. </li><li> As câmaras foram cuidadosamente removido os slides, o excesso de umidade foi apagado e as lâminas foram secas ao ar. </li><li> Uma gota de solução anti Prolongar-fade GOLD (Invitrogen) foi adicionado por poço e lâminas foram montadas (22x50 milímetros lamela) e qualquer excesso de líquido ao redor das bordas do slide foi apagado. </li><li> As lâminas foram mantidas no escuro por mais 30 minutos em temperatura ambiente antes de selar com unha polonês. </li><li> As lâminas foram armazenadas durante a noite a 4 ° C no escuro antes da análise. </li></ol> Microscopia / Análise <ol><li> Zeiss LSM510 Meta Confocal Microscpe utilizados para adquirir imagens utilizando a GFP padrão (para γH2AX - Alexa Fluor 488 cabra anti-IgG de rato) e lasers vermelho distante (para TOPRO-3). Tipicamente, um óleo de 63 x lente objetiva de imersão é usado. Imagens são adquiridas em um padrão Z-série com um tamanho de passo de 0,5 micron. A tamanho do passo de 0,5 m foi escolhida para minimizar a perda de apresentar focos em diferentes planos no núcleo. Durante a análise, planos individuais são deconvoluídos e empilhados para produzir uma imagem máxima projetada para minimizar a sobreposição de focos (Top hat-filtro aplicado). </li><li> Metamorph (dispositivos Molecular, EUA) foi utilizado para analisar o número de focos. O programa quantitates o número de focos em cada célula após o limite tem sido aplicado para excluir fundo. As informações são registradas em uma planilha do Microsoft Excel para análise posterior. </li></ol><img src="/files/ftp_upload/1957/1957fig1.jpg" alt="Figura 1" /> Figura 1. Visualização de imunofluorescência γH2AX focos (verde) em queratinócitos humanos não tratados e em células irradiadas com 2 Gy e incubados por uma hora mais 1 a 37 ° C, 5% CO 2. O DNA foi corado com TOPRO-3 (azul). Imagens foram adquiridas utilizando um microscópio Zeiss LSM 510 Meta Confocal. Bar = 10 mM. e_content &quot;&gt; <img src="/files/ftp_upload/1957/1957fig2.jpg" alt="Figura 2" /> Figura 2. Visualização de imunofluorescência γH2AX focos (verde) em queratinócitos humanos e em células irradiadas com 2 Gy e incubados por uma hora mais 1 a 37 ° C, 5% CO 2. Imagens foram adquiridas utilizando um microscópio Zeiss LSM 510 Meta Confocal utilizando 0,5 m de corte Z-(1-9) para garantir que todos os focos foram adquiridas. As imagens foi então empilhados para quantificação usando Metamorph. O DNA foi corado com TOPRO-3 (azul). O γH2AX empilhados e imagens azuis foram empilhadas para visualização. Bar = 10 mM.

<ol>
	<li>Rogakou, E. P., Boon, C., Redon, C., Bonner, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Megabase+chromatin+domains+involved+in+DNA+double-strand+breaks+in+vivo.">Megabase chromatin domains involved in DNA double-strand breaks in vivo.</a> J. Cell Biol. 146, 905-916 (1999).</li><li>Bonner, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gamma+H2AX+and+cancer.">Gamma H2AX and cancer.</a> Nature Reviews Cancer. 8 (12), 957-967 (2008).</li><li>Savic, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+Dynamic+[gamma]-H2AX+Domains+along+Broken+DNA+Strands+Is+Distinctly+Regulated+by+ATM+and+MDC1+and+Dependent+upon+H2AX+Densities+in+Chromatin.">Formation of Dynamic [gamma]-H2AX Domains along Broken DNA Strands Is Distinctly Regulated by ATM and MDC1 and Dependent upon H2AX Densities in Chromatin.</a> Molecular Cell. 34 (3), 298-310 (2009).</li><li>Downs, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+of+chromatin-modifying+activities+to+phosphorylated+histone+H2A+at+DNA+damage+sites.">Binding of chromatin-modifying activities to phosphorylated histone H2A at DNA damage sites.</a> Mol Cell. 16 (6), 979-990 (2004).</li><li>Chowdhury, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[gamma]-H2AX+Dephosphorylation+by+Protein+Phosphatase+2A+Facilitates+DNA+Double-Strand+Break+Repair.">[gamma]-H2AX Dephosphorylation by Protein Phosphatase 2A Facilitates DNA Double-Strand Break Repair.</a> Molecular Cell. 20 (5), 801-809 (2005).</li><li>Nakada, S., Chen, G., Gingras, A., Durocher, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PP4+is+a+gamma+H2AX+phosphatase+required+for+recovery+from+the+DNA+damage+checkpoint.">PP4 is a gamma H2AX phosphatase required for recovery from the DNA damage checkpoint.</a> EMBO Rep. 9 (10), 1019-1026 (2008).</li><li>Altaf, M., Auger, A., Covic, M., C&#244;t&#233;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connection+between+histone+H2A+variants+and+chromatin+remodeling+complexes.">Connection between histone H2A variants and chromatin remodeling complexes.</a> Biochem Cell Biol. 87 (1), 35-50 (2009).</li><li>Kusch, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetylation+by+Tip60+Is+Required+for+Selective+Histone+Variant+Exchange+at+DNA+Lesions.">Acetylation by Tip60 Is Required for Selective Histone Variant Exchange at DNA Lesions.</a> Science. 306, 2084-2087 (2004).</li><li>Hurlin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progression+of+human+papillomavirus+type+18-immortalized+human+keratinocytes+to+a+malignant+phenotype.">Progression of human papillomavirus type 18-immortalized human keratinocytes to a malignant phenotype.</a> Proc Natl Acad Sci U S A. 88 (2), 570-574 (1991).</li><li>Dickey, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=H2AX:+functional+roles+and+potential+applications.">H2AX: functional roles and potential applications.</a> Chromosoma. , (2009).</li></ol>

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Após a exposição à radiação ionizante (raios-γ), forma γH2AX focos rapidamente e os números de focos atingir um máximo entre 30-60 minutos 2. Portanto, nosso ponto de uma hora o tempo pós-irradiação reflete DSB formação inicial. Temos usado a dose de radiação clinicamente relevantes de 2 Gy para nosso experimento. No entanto, o método pode ser usado para as doses de radiação até 4 Gy para a detecção da formação inicial DSB; sobreposição significativa de focos impede a quantificaçã...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Keratinocyte-Serum Free Medium (K-SFM)</td>
<td>Media</td>
<td>Invitrogen</td>
<td>17005042</td>
<td>Keratinocyte media supplemented with human recombinant Epidermal Growth Factor 1-53 (EGF 1-53), and Bovine Pituitary Extract (BPE).</td>
</tr>
<tr>
<td>Lab Tek lI Chamber Slides (8-well)</td>
<td>Chamber Slides</td>
<td>Nunc Denmark</td>
<td>NUN154534</td>
<td>&nbsp;</td>
<tr>
<td>Coverslips (22x50mm)</td>
<td>Coverslips</td>
<td>Menzel-Gl&auml;ser</td>
<td>CS2250100</td>
<td>&nbsp;</td>
<tr>
<td>Bovine Serum Albumin (BSA)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>A7906</td>
<td>BSA (1%) is used to block any non-specific antibody binding. Primary and secondary antibodies are diluted in BSA.</td>
</tr>
<tr>
<td>PBS (without Ca2+ and Mg2+)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17-517Q</td>
<td>&nbsp;</td>
<tr>
<td>0.5% Trypsin-EDTA x10</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15400-054</td>
<td>Trypsin-EDTA (0.05%) used to detach cells from culture flasks.</td>
</tr>
<tr>
<td>Triton X-100</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>T8787</td>
<td>Triton X-100 (0.1%) used to permeabilise cells.</td>
</tr>
<tr>
<td>Paraformaldehyde</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>158127</td>
<td>Paraformaldehyde (4%) used to fix cells.</td>
</tr>
<tr>
<td>Mouse monoclonal anti-phospho histone-H2AX antibody</td>
<td>Primary Antibody</td>
<td>Millipore</td>
<td>16193</td>
<td>Dilution of primary antibody (1:500), in 1% BSA.</td>
</tr>
<tr>
<td>Alexa Fluor 488 goat anti-mouse IgG (H+L)</td>
<td>Secondary Antibody</td>
<td>Invitrogen</td>
<td>11029</td>
<td>Dilution of secondary antibody (1:500), in 1% BSA.</td>
</tr>
<tr>
<td>TOPRO3</td>
<td>DNA Stain</td>
<td>Invitrogen</td>
<td>T3605</td>
<td>DNA stain commonly used: 4,6-diamidino-2-phenylindole dihydrochloride (DAPI). Can only be used with microscopes with the appropriate excitation laser.</td>
</tr>
<tr>
<td>ProLong Gold</td>
<td>Anti-fade solution</td>
<td>Invitrogen</td>
<td>P36930</td>
<td>Refractive index of 1.42 at 20&deg;C.</td>
</tr>
<tr>
<td>Tissue Culture Flask, Vented Cap</td>
<td>Culture Flask</td>
<td>BD Falcon</td>
<td>353112</td>
<td>&nbsp;</td>
<tr>
<td>Tissue Culture Dish (150x25mm)</td>
<td>Petridish</td>
<td>BD Falcon</td>
<td>353025</td>
<td>&nbsp;</td>
<tr>
<td>Coplin Jar, glass</td>
<td>&nbsp;</td>
<td>Grale Scientific P/L</td>
<td>1771-OG</td>
<td>&nbsp;</td>
<tr>
<td>Staining Trough</td>
<td>&nbsp;</td>
<td>Grale Scientific P/L</td>
<td>V1991.99</td>
<td>&nbsp;</td>
<tr>
<td>Gammacell 1000 Elite Irradiator</td>
<td>Gamma Irradiator</td>
<td>Nordion International Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Zeiss LSM 510 Meta Confocal</td>
<td>Confocal Microscope</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Metamorph</td>
<td>Software for Imaging analysis</td>
<td>Molecular Devices, USA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

quantification of &gamma;h2ax foci in response to ionising radiation

DNA dupla fita-breaks (LAP), que são induzidos por qualquer processos metabólicos endógenos ou por fontes exógenas, são uma das lesões DNA mais críticos com relação à sobrevivência e preservação da integridade genômica. Uma resposta rápida à indução de LAP é fosforilação da variante histona H2A, H2AX, na serina-139 resíduos, na altamente conservada motivo SQEY C-terminal, formando γH2AX<sup&gt; 1</sup&gt;. Após a indução de LAP, H2AX é rapidamente fosforilada pela fosfatidil-inosito 3-quinase (Pikk) família de proteínas, ataxia telangiectasia mutantes (ATM), subunidade proteína-quinase DNA catalítico e ATM e Rad3 relacionados (ATR)<sup&gt; 2</sup&gt;. Tipicamente, apenas alguns pares-base (pb) estão implicados em uma DSB, no entanto, não há amplificação de sinal significativo, dada a importância das modificações da cromatina na sinalização de danos ao DNA e reparo. Fosforilação de H2AX mediada predominantemente por ATM espalha para áreas adjacentes da cromatina, afetando aproximadamente 0,03% do total celulares H2AX por DSB<sup&gt; 2,3</sup&gt;. Isso corresponde a fosforilação de aproximadamente 2000 moléculas H2AX abrangendo ~ 2 Mbp regiões da cromatina em torno do local do DSB e resulta na formação de discretos focos γH2AX que podem ser facilmente visualizados e quantificados por microscopia de imunofluorescência<sup&gt; 2</sup&gt;. A perda de γH2AX no DSB reflete reparação, no entanto, há alguma controvérsia quanto ao que define o reparo completo da LAP, tem sido proposto que a recombinação de dois filamentos de DNA é adequada no entanto, também tem sido sugerido que restabelecimento do estado de compactação da cromatina originais é necessário<sup&gt; 08/04</sup&gt;. O desaparecimento de γH2AX envolve pelo menos em parte, pela desfosforilação fosfatases, fosfatase e fosfatase 2A 4C<sup&gt; 5,6</sup&gt;. Além disso, a remoção de γH2AX pela redistribuição envolvendo troca de histonas com H2A.Z tem sido implicado<sup&gt; 7,8</sup&gt;. Importante, a análise quantitativa de γH2AX focos levou a uma ampla gama de aplicações na pesquisa médica e nuclear. Aqui, nós demonstrar o método mais comumente usado de imunofluorescência para avaliação de danos no DNA inicial de detecção e quantificação de γH2AX focos em γ-irradiados aderente queratinócitos humanos<sup&gt; 9</sup&gt;.

Watch this Scientific Journal Video about Quantification of Î³H2AX Foci in Response to Ionising Radiation at JoVE.com

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

Quantificação de DNA dupla fita-estrias usando formação γH2AX como um marcador molecular tornou-se uma ferramenta valiosa na biologia da radiação. Aqui mostramos o uso de um teste de imunofluorescência para a quantificação dos focos γH2AX após a exposição das células à radiação.

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 ...

quantification-of-h2ax-foci-in-response-to-ionising-radiation

Research

JoVE Journal

Biology

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

20.3K visualizações.  The Alfred Medical Research and Education Precinct. Quantificação de DNA dupla fita-estrias usando formação γH2AX como um marcador molecular tornou-se uma ferramenta valiosa na biologia da radiação. Aqui mostramos o uso de um teste de imunofluorescência para a quantificação dos focos γH2AX após a exposição das células à radiação.

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

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 &amp;#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) ...

Biologia

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 &amp;#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 ...

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

Duas e três dimensões imagens de células vivas de Proteínas de DNA resposta a danos

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

Measuring Glutathione-induced Feeding Response in Hydra

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. The movement of prey organisms causes mechanosensory discharge of the stinging cells called nematocysts from hydra, which are inserted into the prey. The feeding response in hydra, which includes curling of the tentacles to bring the prey towards the mouth, opening of the mouth and consequent engulfing of the prey, is triggered by GSH present in the fluid released from the injured prey. To be able to identify the molecular mechanism of the feeding response in hydra which is unknown to date, it is necessary to establish an assay to measure the feeding response. Here, we describe a simple method for the quantitation of the feeding response in which the distance between the apical end of the tentacle and mouth of hydra is measured and the ratio of such distance before and after the addition of GSH is determined. The ratio, called the relative tentacle spread, was found to give a measure of the feeding response. This assay was validated using a starvation model in which starved hydra show an enhanced feeding response in comparison with daily fed hydra.

Here we describe a simple assay for the quantification of the feeding response in hydra induced by the reduced form of glutathione. This assay relies on measuring the distance between the apical end of the tentacle and mouth of hydra.

Medindo alimentação resposta induzida por glutationa em Hydra

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. ...

Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic knockdown system, we showed novel functions for HDAC1,2 in replication fork progression and nascent chromatin maintenance in mammalian cells. Additionally, we used a BrdU-ChIP-Slot-Western technique that combines chromatin immunoprecipitation (ChIP) of bromo-deoxyuridine (BrdU)-labeled DNA with slot blot and Western analyses to quantitatively measure proteins or histone modification associated with nascent DNA.
Actively dividing cells were treated with HDAC1,2 selective inhibitor or transfected with siRNAs against Hdac1 and Hdac2 and then newly synthesized DNA was labeled with the thymidine analog bromodeoxyuridine (BrdU). The BrdU labeling was done at a time point when there was no significant cell cycle arrest or apoptosis due to the loss of HDAC1,2 functions. Following labeling of cells with BrdU, chromatin immunoprecipitation (ChIP) of histone acetylation marks or the chromatin-remodeler was performed with specific antibodies. BrdU-labeled input DNA and the immunoprecipitated (or ChIPed) DNA was then spotted onto a membrane using the slot blot technique and immobilized using UV. The amount of nascent DNA in each slot was then quantitatively assessed using Western analysis with an anti-BrdU antibody. The effect of loss of HDAC1,2 functions on the levels of newly synthesized DNA-associated histone acetylation marks and chromatin remodeler was then determined by normalizing the BrdU-ChIP signal obtained from the treated samples to the control samples.

In this protocol, we describe a novel BrdU-ChIP-Slot-Western technique to examine proteins and histone modifications associated with newly synthesized or nascent DNA.

Exame de proteínas ligadas ao DNA em células de mamíferos Nascente usar a técnica de-BrdU-chip-Slot Ocidental

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

Quantificação de alta resolução Celulose cristalina Acumulação em<em&gt; Arabidopsis</em&gt; Raízes para monitorar celular específico do tecido da parede Modificações

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

Methods to Classify Cytoplasmic Foci as Mammalian Stress Granules

Cells are often challenged by sudden environmental changes. Stress Granules (SGs), cytoplasmic ribonucleoprotein complexes that form in cells exposed to stress conditions, are implicated in various aspects of cell metabolism and survival. SGs modulate cellular signaling pathways, post-transcriptional gene expression, and stress response programs. The formation of these mRNA-containing granules is directly connected to cellular translation. SG assembly is triggered by inhibited translation initiation, and SG disassembly is promoted by translation activation or by inhibited translation elongation. This relationship is further highlighted by SG composition. Core SG components are stalled translation pre-initiation complexes, mRNA, and selected RNA-binding Proteins (RBPs). The purpose of SG assembly is to conserve cellular energy by sequestering translationally stalled housekeeping mRNAs, allowing for the enhanced translation of stress-responsive proteins. In addition to the core constituents, such as stalled translation preinitiation complexes, SGs contain a plethora of other proteins and signaling molecules. Defects in SG formation can impair cellular adaptation to stress and can thus promote cell death. SGs and similar RNA-containing granules have been linked to a number of human diseases, including neurodegenerative disorders and cancer, leading to the recent interest in classifying and defining RNA granule subtypes. This protocol describes assays to characterize and quantify mammalian SGs.

Stress Granules (SGs) are nonmembranous cytoplasmic structures that form in cells exposed to a variety of stresses. SGs contain mRNAs, RNA-binding proteins, small ribosomal subunits, translation-related factors, and various cell signaling proteins. This protocol describes a workflow that uses several experimental approaches to detect, characterize, and quantify bona fide SGs.

Métodos para classificar os focos citoplasmáticos como grânulos de estresse de mamíferos

Cells are often challenged by sudden environmental changes. Stress Granules (SGs), cytoplasmic ribonucleoprotein complexes that form in cells exposed to ...

Intestinal Epithelial Regeneration in Response to Ionizing Irradiation

The intestinal epithelium consists of a single layer of cells yet contains multiple types of terminally differentiated cells, which are generated by the active proliferation of intestinal stem cells located at the bottom of intestinal crypts. However, during events of acute intestinal injury, these active intestinal stem cells undergo cell death. Gamma irradiation is a widely used colorectal cancer treatment, which, while therapeutically efficacious, has the side effect of depleting the active stem cell pool. Indeed, patients frequently experience gastrointestinal radiation syndrome while undergoing radiotherapy, in part due to active stem cell depletion. The loss of active intestinal stem cells in intestinal crypts activates a pool of typically quiescent reserve intestinal stem cells and induces dedifferentiation of secretory and enterocyte precursor cells. If not for these cells, the intestinal epithelium would lack the ability to recover from radiotherapy and other such major tissue insults. New advances in lineage-tracing technologies allow tracking of the activation, differentiation, and migration of cells during regeneration and have been successfully employed for studying this in the gut. This study aims to depict a method for the analysis of cells within the mouse intestinal epithelium following radiation injury.

The gastrointestinal tract is one of the most sensitive organs to injury upon radiotherapeutic cancer treatments. It is simultaneously an organ system with one of the highest regenerative capacities following such insults. The presented protocol describes an efficient method to study the regenerative capacity of the intestinal epithelium.

Regeneração Epitelial Intestinal em Resposta à Irradiação Ionizante

The intestinal epithelium consists of a single layer of cells yet contains multiple types of terminally differentiated cells, which are generated by the ...

Quantificação da infiltração gordurosa do músculo esquelético via descelularização

Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration

Fatty infiltration is the accumulation of adipocytes between myofibers in skeletal muscle and is a prominent feature of many myopathies, metabolic disorders, and dystrophies. Clinically in human populations, fatty infiltration is assessed using noninvasive methods, including computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound (US). Although some studies have used CT or MRI to quantify fatty infiltration in mouse muscle, costs and insufficient spatial resolution remain challenging. Other small animal methods utilize histology to visualize individual adipocytes; however, this methodology suffers from sampling bias in heterogeneous pathology. This protocol describes the methodology to qualitatively view and quantitatively measure fatty infiltration comprehensively throughout intact mouse muscle and at the level of individual adipocytes using decellularization. The protocol is not limited to specific muscles or specific species and can be extended to human biopsy. Additionally, gross qualitative and quantitative assessments can be made with standard laboratory equipment for little cost, making this procedure more accessible across research laboratories.

Autor em destaque: Quantificação baseada em descelularização da infiltração gordurosa do músculo esquelético  

The present study describes decellularization-based methodologies for visualizing and quantifying intramuscular adipose tissue (IMAT) deposition through intact muscle volume, as well as quantifying metrics of individual adipocytes that comprise IMAT.

Methods Article

Quantificação de γH2AX Focos em resposta à radiação ionizante