Name: quantification of &gamma;h2ax foci in response to ionising radiation
Uploaded: 2010-04-06T19:35:00
Description: Watch this Scientific Journal Video about Quantification of ÃŽÂ³H2AX Foci in Response to Ionising Radiation at JoVE.com

De steun van het Australian Institute of Nuclear Science and Engineering wordt erkend. TCK was de ontvanger van AINSE awards. Epigenomisch Medicine Lab wordt ondersteund door de National Health en Medical Research Council van Australië (566.559). LM wordt ondersteund door Melbourne Research (Universiteit van Melbourne) en Biomedical Imaging CRC aanvullende beurzen. De ondersteuning van Monash Micro Imaging (Drs Stephen Cody en Iska Carmichael) was van onschatbare waarde voor dit werk.

<html> Celpreparaat <ol><li> Humane keratinocyten (FEP-1811) werden gekweekt in keratinocyten-serum vrij medium (K-SFM; Invitrogen), aangevuld met epidermale groeifactor, runder-hypofyse-extract en 20 ug / ml gentamycine, bij 37 ° C en 5% CO 2. </li><li> Een enkele celsuspensie werd bereid door het losmaken met trypsine-EDTA (0,05% v / v) </li><li> Cellen werden gezaaid in 8-goed Lab Tek II microchamber dia&#39;s (10.000 cellen / putje) en dia&#39;s werden gedurende 3 dagen bij 37 ° C en 5% CO 2.</sub...

<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>

Na blootstelling aan ioniserende straling (γ-stralen), γH2AX foci vorm snel en foci nummers oplopen tot tussen de 30-60 minuten 2. Daarom is onze 1 uur na de bestraling tijdstip weerspiegelt de initiële vorming van DSB. Wij hebben gebruik gemaakt van de klinisch relevante stralingsdosis van 2 Gy voor ons experiment. Toch kan de methode worden gebruikt voor straling doses tot 4 Gy voor de detectie van de eerste DSB formatie; belangrijke overlapping van foci sluit accurate kwantificering bij hogere doseringe...

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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 dubbel-breuken (DSB&#39;s), die worden veroorzaakt door een endogene metabole processen of door exogene bronnen, zijn een van de meest kritische DNA-laesies met betrekking tot de overleving en behoud van de genomische integriteit. Een vroege reactie op de inductie van DSB is fosforylatie van het H2A histon-variant, H2AX, op de serine-139 residu, in de zeer geconserveerd C-terminale SQEY motief, de vorming van γH2AX<sup&gt; 1</sup&gt;. Na inductie van DSB&#39;s, is H2AX snel gefosforyleerd door het fosfatidyl-inosito 3-kinase (Pikk) familie van eiwitten, ataxie telangiectasia gemuteerd (ATM), DNA-eiwit kinase katalytische subeenheid en ATM en RAD3-gerelateerde (ATR)<sup&gt; 2</sup&gt;. Gewoonlijk worden slechts een paar baseparen (bp) betrokken bij een DSB, is er echter aanzienlijke signaalversterking, gezien het belang van chromatine modificaties in DNA-schade signaleren en repareren. Fosforylatie van H2AX gemedieerd hoofdzakelijk door ATM zich uitbreidt naar aangrenzende gebieden van chromatine, die ongeveer 0,03% van het totale cellulaire H2AX per DSB<sup&gt; 2,3</sup&gt;. Dit komt overeen met fosforylering van ongeveer 2000 H2AX moleculen verspreid over ~ 2 Mbp regio&#39;s van chromatine rond de site van de DSB en resulteert in de vorming van discrete γH2AX haarden, die gemakkelijk kunnen worden gevisualiseerd en gekwantificeerd door immunofluorescentie microscopie<sup&gt; 2</sup&gt;. Het verlies van γH2AX bij DSB weerspiegelt reparatie, maar er is enige controverse over wat definieert volledige reparatie van DSB, het is voorgesteld dat hereniging van beide strengen van het DNA is echter voldoende, is het ook gesuggereerd dat opnieuw het de originele chromatine toestand van verdichting is noodzakelijk<sup&gt; 4-8</sup&gt;. De verdwijning van γH2AX omvat ten minste gedeeltelijk, defosforylering door fosfatasen, fosfatase 2A en fosfatase 4C<sup&gt; 5,6</sup&gt;. Verder is het verwijderen van γH2AX door herverdeling met histon-uitwisseling met H2A.Z zijn betrokken<sup&gt; 7,8</sup&gt;. Belangrijker is dat de kwantitatieve analyse van γH2AX foci geleid tot een breed scala aan toepassingen in de medische en nucleair onderzoek. Hier tonen we de meest gebruikte immunofluorescentie methode voor de evaluatie van de eerste DNA-schade door detectie en kwantificatie van γH2AX foci in γ-bestraalde aanhanger humane keratinocyten<sup&gt; 9</sup&gt;.

Watch this Scientific Journal Video about Quantification of ÃŽÂ³H2AX Foci in Response to Ionising Radiation at JoVE.com

Quantification of ÃƒÅ½Ã‚Â³H2AX Foci in Response to Ionising Radiation

Kwantificering van DNA dubbel-strengs strepen met behulp van γH2AX vorming als een moleculaire marker is uitgegroeid tot een onmisbaar instrument in straling biologie. Hier laten we zien het gebruik van een immunofluorescentie assay voor de kwantificering van γH2AX foci na blootstelling van cellen aan straling.

Kwantificering van γH2AX Foci in reactie op ioniserende straling

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

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