Name: evaluation of the spatial distribution of &gamma;h2ax following ionizing radiation
Uploaded: 2010-08-07T17:00:00
Description: Watch this Scientific Journal Video about Evaluation of the Spatial Distribution of Î³H2AX following Ionizing Radiation Video at JoVE.com

The support of the Australian Institute of Nuclear Science and Engineering is acknowledged. TCK was the recipient of AINSE awards. Epigenomic Medicine Lab is supported by the National Health and Medical Research Council of Australia (566559). This work is funded by the CRC for Biomedical Imaging Development Ltd, established and supported under the Australian Government s Cooperative Research Centres (CRC) program. LM is supported by Melbourne Research (University of Melbourne) and Biomedical Imaging CRC supplementary scholarships. The support of Monash Micro Imaging (Drs Stephen Cody and I&#347;ka Carmichael) was invaluable for this work.

Cell preparation
<ol>
<li>	Human erythroleukemic K562 cells are grown in RPMI-1640 medium supplemented with 10% (v/v) fetal bovine serum and 20 mg/ml gentamicin in a humidified 5% CO2 environment at 37&deg;C.</li>
<li>	Approximately 18 hours prior to staining wash exponentially growing cells (optimal at 5 x 105 cells/ml) with phosphate buffered saline (PBS), resuspend in fresh media and return to 37&deg;C, 5% CO2.</li>
<li>	Wash the cells twice with PBS by centrifugation at approxim...

<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 (5), 905-916 (1999).</li><li>Rogakou, E. P., Pilch, D. R., Orr, A. H., Ivanova, V. S., 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=DNA+double-stranded+breaks+induce+histone+H2AX+phosphorylation+on+serine+139.">DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139.</a> J Biol Chem. 273 (10), 5858-5868 (1998).</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=.">.</a> Nat Rev Cancer. 8 (12), 957-967 (2008).</li><li>Dickey, J. S. <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. 118 (6), 683-692 (2009).</li><li>Kim, J. A., Kruhlak, M., Dotiwala, F., Nussenzweig, A., Haber, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterochromatin+is+refractory+to+gamma-H2AX+modification+in+yeast+and+mammals.">Heterochromatin is refractory to gamma-H2AX modification in yeast and mammals.</a> J Cell Biol. 178 (2), 209-218 (2007).</li><li>Cowell, I. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=gammaH2AX+foci+form+preferentially+in+euchromatin+after+ionising-radiation.">gammaH2AX foci form preferentially in euchromatin after ionising-radiation.</a> PLoS One. 2 (10), e1057-e1057 (2007).</li><li>Kinner, A., Wu, W., Staudt, C., &amp;amp, I. l. i. a. k. i. s., G, . <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+in+recognition+and+signaling+of+DNA+double-strand+breaks+in+the+context+of+chromatin.">Gamma-H2AX in recognition and signaling of DNA double-strand breaks in the context of chromatin.</a> Nucleic Acids Res. 36 (17), 5678-5694 (2008).</li><li>Vasireddy, R. S., Karagiannis, T. C., El-Osta, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=gamma-radiation-induced+gammaH2AX+formation+occurs+preferentially+in+actively+transcribing+euchromatic+loci.">gamma-radiation-induced gammaH2AX formation occurs preferentially in actively transcribing euchromatic loci.</a> Cell Mol Life Sci. 67 (2), 291-294 (2010).</li><li>Goodarzi, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATM+signaling+facilitates+repair+of+DNA+double-strand+breaks+associated+with+heterochromatin.">ATM signaling facilitates repair of DNA double-strand breaks associated with heterochromatin.</a> Mol Cell. 31 (2), 167-177 (2008).</li></ol>

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		<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>Roswell Park Memorial Institute -1640 (RPMI-1640)</td>
<td>Growth medium</td>
<td>Invitrogen</td>
<td>22400071</td>
<td>RPMI-1640, pH 7.4 medium supplemented with 10% (v/v) fetal bovine, 2mM L-glutamine, 20&#x03BC;g/ml gentamicin, 20mM (HEPES) N-2-Hydroxyethylpiperazine-N&#x2019;-2-Ethanesulfonic Acid</td>
</tr>
<tr>
<td>Fetal Bovine Serum (FBS)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>F2442</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>Trypan blue</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>T6146</td>
<td>Used to distinguish between live and dead cells.</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, USA</td>
<td>16193</td>
<td>Dilution of primary antibody (1:500), in 1% BSA.</td>
</tr>
<tr>
<td>HistoneH3 (Mono-methyl K4) </td>
<td>Primary Antibody</td>
<td>Abcam, Cambridge, UK</td>
<td>AB8895</td>
<td>&nbsp;</td>
<tr>
<td>Alexa Fluor 488 goat anti-mouse IgG (H+L)</td>
<td>Secondary Antibody</td>
<td>Invitrogen, USA</td>
<td>11029</td>
<td>Dilution of secondary antibody (1:500), in 1% BSA.</td>
</tr>
<tr>
<td>Alexa Fluor 546 goat anti-rabbit IgG (H+L)</td>
<td>Secondary Antibody</td>
<td>Invitrogen, USA</td>
<td>11035</td>
<td>&nbsp;</td>
<tr>
<td>TOPRO3</td>
<td>DNA Stain</td>
<td>Invitrogen, USA</td>
<td>T3605</td>
<td>TOPRO3 is a DNA dye with an Abs/Em of 642/661 nm. DAPI could be used if the confocal microscope is equipped with a 405 nm laser. </td>
</tr>
<tr>
<td>ProLong Gold </td>
<td>Anti-fade solution</td>
<td>Invitrogen</td>
<td>P36930</td>
<td>This glycerol based mounting medium must be used with an oil based lense that matches its refractive index.</td>
</tr>
<tr>
<td>Polylysine slides</td>
<td>&nbsp;</td>
<td>Menzel-Gl&#228;ser, Germany</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Coverslips (22x50mm)</td>
<td>Coverslips</td>
<td>Menzel-Gl&#228;ser, Germany</td>
<td>CS2250100</td>
<td>&nbsp;</td>
<tr>
<td>Tissue Culture Flask, Vented Cap</td>
<td>Culture Flask</td>
<td>BD Falcon</td>
<td>353112</td>
<td>&nbsp;</td>
<tr>
<td>Shandon Cytospin 4</td>
<td>&nbsp;</td>
<td>ThermoElectron Corp</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cytofunnels</td>
<td>&nbsp;</td>
<td>Shandon, Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Filter Cards </td>
<td>&nbsp;</td>
<td>Shandon, Inc</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>PAP Pen</td>
<td>&nbsp;</td>
<td>Zymed Laboratories, Invitrogen</td>
<td> 008877</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>Equipped with 3 lasers: 488 nm, 543 nm and 633 nm.</td>
</tr>
<tr>
<td>Metamorph</td>
<td>Software for Imaging analysis</td>
<td>Molecular Devices, USA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
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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.

Watch this Scientific Journal Video about Evaluation of the Spatial Distribution of Î³H2AX following Ionizing Radiation Video at JoVE.com

Evaluation of the Spatial Distribution of Î³H2AX following Ionizing Radiation Video (Video) | JoVE

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.

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 ...

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