Name: cell cycle-specific measurement of &#947;h2ax and apoptosis after genotoxic stress by flow cytometry
Uploaded: 2019-09-01T14:00:00
Description: Watch this Scientific Journal Video about Cell Cycle-specific Measurement of ÃŽÂ³H2AX and Apoptosis After Genotoxic Stress by Flow Cytometry at JoVE.com

We thank the Flow Cytometry Facility team at the German Cancer Research Center (DKFZ) for their support.

1. Preparation
<ol>
	<li>Prepare &#8805;1 x 105 cells/sample in any type of culture vessel as starting material.
	<ol>
		<li>For example, conduct a time-course experiment after exposure of U87 glioblastoma cells to ionizing radiation: Irradiate sub-confluent U87 cells in T25 flasks in triplicates for each time point. Choose early time-points (15 min up to 8 h after irradiation) to follow the kinetics of DSB repair (&#947;H2AX level) and late time points (24 h up to 96 h) to assess residual D...

<ol>
	<li>Jensen, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+non-small+cell+lung+cancer+with+intensity-modulated+radiation+therapy+in+combination+with+cetuximab:+the+NEAR+protocol+(NCT00115518).">Treatment of non-small cell lung cancer with intensity-modulated radiation therapy in combination with cetuximab: the NEAR protocol (NCT00115518).</a> BMC Cancer. 6, 122 (2006).</li><li>Oertel, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Glioblastoma+and+Carcinoma+Xenograft+Tumors+Treated+by+Combined+Radiation+and+Imatinib+(Gleevec).">Human Glioblastoma and Carcinoma Xenograft Tumors Treated by Combined Radiation and Imatinib (Gleevec).</a> Strahlentherapie und Onkologie. 182 (7), 400-407 (2006).</li><li>Timke, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+of+Vascular+Endothelial+Growth+Factor+Receptor/Platelet-Derived+Growth+Factor+Receptor+Inhibition+Markedly+Improves+Radiation+Tumor+Therapy.">Combination of Vascular Endothelial Growth Factor Receptor/Platelet-Derived Growth Factor Receptor Inhibition Markedly Improves Radiation Tumor Therapy.</a> Clinical Cancer Research. 14 (7), 2210-2219 (2008).</li><li>Zhang, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trimodal+glioblastoma+treatment+consisting+of+concurrent+radiotherapy,+temozolomide,+and+the+novel+TGF-&#946;+receptor+I+kinase+inhibitor+LY2109761.">Trimodal glioblastoma treatment consisting of concurrent radiotherapy, temozolomide, and the novel TGF-&#946; receptor I kinase inhibitor LY2109761.</a> Neoplasia. 13 (6), 537-549 (2011).</li><li>Blattmann, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suberoylanilide+hydroxamic+acid+affects+expression+in+osteosarcoma,+atypical+teratoid+rhabdoid+tumor+and+normal+tissue+cell+lines+after+irradiation.">Suberoylanilide hydroxamic acid affects expression in osteosarcoma, atypical teratoid rhabdoid tumor and normal tissue cell lines after irradiation.</a> Strahlentherapie und Onkologie. 188 (2), 168-176 (2012).</li><li>Hoeijmakers, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+Damage,+Aging,+and+Cancer.">DNA Damage, Aging, and Cancer.</a> New England Journal of Medicine. 361 (15), 1475-1485 (2015).</li><li>Rodgers, K., McVey, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Error-Prone+Repair+of+DNA+Double-Strand+Breaks.">Error-Prone Repair of DNA Double-Strand Breaks.</a> Journal of Cellular Physiology. 231 (1), 15-24 (2016).</li><li>Ciccia, A., Elledge, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+DNA+Damage+Response:+Making+It+Safe+to+Play+with+Knives.">The DNA Damage Response: Making It Safe to Play with Knives.</a> Molecular Cell. 40 (2), 179-204 (2010).</li><li>Rothkamm, K., Kr&#252;ger, I., Thompson, L. H., L&#246;brich, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathways+of+DNA+double-strand+break+repair+during+the+mammalian+cell+cycle.">Pathways of DNA double-strand break repair during the mammalian cell cycle.</a> Molecular and Cellular Biology. 23 (16), 5706-5715 (2003).</li><li>Escribano-D&#237;az, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Cell+Cycle-Dependent+Regulatory+Circuit+Composed+of+53BP1-RIF1+and+BRCA1-CtIP+Controls+DNA+Repair+Pathway+Choice.">A Cell Cycle-Dependent Regulatory Circuit Composed of 53BP1-RIF1 and BRCA1-CtIP Controls DNA Repair Pathway Choice.</a> Molecular Cell. 49 (5), 872-883 (2013).</li><li>Bakr, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+crosstalk+between+DNA+damage+response+proteins+53BP1+and+BRCA1+regulates+double+strand+break+repair+choice.">Functional crosstalk between DNA damage response proteins 53BP1 and BRCA1 regulates double strand break repair choice.</a> Radiotherapy and Oncology. 119 (2), 276-281 (2015).</li><li>Mladenov, E., Magin, S., Soni, A., Iliakis, G. <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-strand-break+repair+in+higher+eukaryotes+and+its+role+in+genomic+instability+and+cancer:+Cell+cycle+and+proliferation-dependent+regulation.">DNA double-strand-break repair in higher eukaryotes and its role in genomic instability and cancer: Cell cycle and proliferation-dependent regulation.</a> Seminars in Cancer Biology. 37-38, 51-64 (2016).</li><li>Lopez Perez, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+damage+response+of+clinical+carbon+ion+versus+photon+radiation+in+human+glioblastoma+cells.">DNA damage response of clinical carbon ion versus photon radiation in human glioblastoma cells.</a> Radiotherapy and Oncology. 133, 77-86 (2019).</li><li>Oertel, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+of+suberoylanilide+hydroxamic+acid+with+heavy+ion+therapy+shows+promising+effects+in+infantile+sarcoma+cell+lines.">Combination of suberoylanilide hydroxamic acid with heavy ion therapy shows promising effects in infantile sarcoma cell lines.</a> Radiation oncology. 6 (1), 119 (2011).</li><li>Blattmann, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suberoylanilide+hydroxamic+acid+affects+&#947;H2AX+expression+in+osteosarcoma,+atypical+teratoid+rhabdoid+tumor+and+normal+tissue+cell+lines+after+irradiation.">Suberoylanilide hydroxamic acid affects &#947;H2AX expression in osteosarcoma, atypical teratoid rhabdoid tumor and normal tissue cell lines after irradiation.</a> Strahlentherapie und Onkologie. 188 (2), 168-176 (2012).</li><li>Thiemann, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+efficacy+of+the+histone+deacetylase+inhibitor+suberoylanilide+hydroxamic+acid+in+combination+with+radiotherapy+in+a+malignant+rhabdoid+tumor+mouse+model.">In vivo efficacy of the histone deacetylase inhibitor suberoylanilide hydroxamic acid in combination with radiotherapy in a malignant rhabdoid tumor mouse model.</a> Radiation Oncology. 7 (1), 52 (2012).</li><li>Nicolay, N. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+retain+their+defining+stem+cell+characteristics+after+exposure+to+ionizing+radiation.">Mesenchymal stem cells retain their defining stem cell characteristics after exposure to ionizing radiation.</a> International Journal of Radiation Oncology Biology Physics. 87 (5), 1171-1178 (2013).</li><li>Nicolay, N. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+are+resistant+to+carbon+ion+radiotherapy.">Mesenchymal stem cells are resistant to carbon ion radiotherapy.</a> Oncotarget. 6 (4), 2076-2087 (2015).</li><li>Nicolay, N. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+exhibit+resistance+to+topoisomerase+inhibition.">Mesenchymal stem cells exhibit resistance to topoisomerase inhibition.</a> Cancer letters. 374 (1), 75-84 (2016).</li><li>Nicolay, N. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+maintain+their+defining+stem+cell+characteristics+after+treatment+with+cisplatin.">Mesenchymal stem cells maintain their defining stem cell characteristics after treatment with cisplatin.</a> Scientific Reports. 6, 20035 (2016).</li><li>Nicolay, N. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+are+sensitive+to+bleomycin+treatment.">Mesenchymal stem cells are sensitive to bleomycin treatment.</a> Scientific Reports. 6, 26645 (2016).</li><li>R&#252;hle, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cisplatin+radiosensitizes+radioresistant+human+mesenchymal+stem+cells.">Cisplatin radiosensitizes radioresistant human mesenchymal stem cells.</a> Oncotarget. 8 (50), 87809-87820 (2017).</li><li>M&#252;nz, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+mesenchymal+stem+cells+lose+their+functional+properties+after+paclitaxel+treatment.">Human mesenchymal stem cells lose their functional properties after paclitaxel treatment.</a> Scientific Reports. 8 (1), 312 (2018).</li><li>R&#252;hle, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Radiation+Resistance+of+Human+Multipotent+Mesenchymal+Stromal+Cells+Is+Independent+of+Their+Tissue+of+Origin.">The Radiation Resistance of Human Multipotent Mesenchymal Stromal Cells Is Independent of Their Tissue of Origin.</a> International Journal of Radiation Oncology Biology Physics. 100 (5), 1259-1269 (2018).</li><li>Dean, P. N., Jett, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mathematical+analysis+of+DNA+distributions+derived+from+flow+microfluorometry.">Mathematical analysis of DNA distributions derived from flow microfluorometry.</a> Journal of Cell Biology. 60 (2), 523-527 (1974).</li><li>Fox, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+for+the+computer+analysis+of+synchronous+DNA+distributions+obtained+by+flow+cytometry.">A model for the computer analysis of synchronous DNA distributions obtained by flow cytometry.</a> Cytometry. 1 (1), 71-77 (1980).</li><li>Costes, S. V., Boissi&#232;re, A., Ravani, S., Romano, R., Parvin, B., Barcellos-Hoff, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+features+that+discriminate+between+foci+induced+by+high-+and+low-LET+radiation+in+human+fibroblasts.">Imaging features that discriminate between foci induced by high- and low-LET radiation in human fibroblasts.</a> Radiation Research. 165 (5), 505-515 (2006).</li><li>Meyer, B., Voss, K. -. O., Tobias, F., Jakob, B., Durante, M., Taucher-Scholz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clustered+DNA+damage+induces+pan-nuclear+H2AX+phosphorylation+mediated+by+ATM+and+DNA-PK.">Clustered DNA damage induces pan-nuclear H2AX phosphorylation mediated by ATM and DNA-PK.</a> Nucleic Acids Research. 41 (12), 6109-6118 (2013).</li><li>Lopez Perez, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superresolution+light+microscopy+shows+nanostructure+of+carbon+ion+radiation-induced+DNA+double-strand+break+repair+foci.">Superresolution light microscopy shows nanostructure of carbon ion radiation-induced DNA double-strand break repair foci.</a> FASEB. 30 (8), 2767-2776 (2016).</li><li>Schipler, A., Iliakis, G. <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-strand-break+complexity+levels+and+their+possible+contributions+to+the+probability+for+error-prone+processing+and+repair+pathway+choice.">DNA double-strand-break complexity levels and their possible contributions to the probability for error-prone processing and repair pathway choice.</a> Nucleic Acids Research. 41 (16), 7589-7605 (2013).</li><li>Stenerlow, B., Hoglund, E., Carlsson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+fragmentation+by+charged+particle+tracks.">DNA fragmentation by charged particle tracks.</a> Advances in Space Research. 30 (4), 859-863 (2002).</li><li>Friedland, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+track-structure+based+evaluation+of+DNA+damage+by+light+ions+from+radiotherapy-relevant+energies+down+to+stopping.">Comprehensive track-structure based evaluation of DNA damage by light ions from radiotherapy-relevant energies down to stopping.</a> Scientific Reports. 7, 45161 (2017).</li><li>Pang, D., Chasovskikh, S., Rodgers, J. E., Dritschilo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+DNA+Fragments+Are+a+Hallmark+of+Heavy+Charged-Particle+Irradiation+and+May+Underlie+Their+Greater+Therapeutic+Efficacy.">Short DNA Fragments Are a Hallmark of Heavy Charged-Particle Irradiation and May Underlie Their Greater Therapeutic Efficacy.</a> Frontiers in Oncology. 6, 130 (2016).</li><li>B&#246;cker, W., Iliakis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computational+Methods+for+analysis+of+foci:+validation+for+radiation-induced+gamma-H2AX+foci+in+human+cells.">Computational Methods for analysis of foci: validation for radiation-induced gamma-H2AX foci in human cells.</a> Radiation Research. 165 (1), 113-124 (2006).</li><li>L&#246;brich, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#947;H2AX+foci+analysis+for+monitoring+DNA+double-strand+break+repair:+Strengths,+limitations+and+optimization.">&#947;H2AX foci analysis for monitoring DNA double-strand break repair: Strengths, limitations and optimization.</a> Cell Cycle. 9 (4), 662-669 (2010).</li></ol>

The featured method is easy to use and offers a fast, accurate and reproducible measurement of the DNA damage response including double-strand break (DSB) induction and repair, cell cycle effects and apoptotic cell death. The combination of these endpoints provides a more complete picture of their interrelations than individual assays. The method can be widely applied as a comprehensive genotoxicity assay in the fields of radiation biology, therapy and protection, and more generally in oncology (e.g., for environmental r...

The goal of oncology is to kill or to inactivate cancer cells without harming normal cells. Many therapies either directly or indirectly induce genotoxic stress in cancer cells, but also to some extend in normal cells. Chemotherapy or targeted drugs are often combined with radiotherapy to enhance the radiosensitivity of the irradiated tumor1,2,3,4,5, which allows for a reduction of the radiation dose to minimize normal tissue damage.
Ionizing radiation and other genotoxic agents induce different kinds of DNA damage, including base modifications, strand crosslinks and single- or double-strand breaks. DNA double-strand breaks (DSBs) are the most serious DNA lesions and their induction is key to the cell killing effect of ionizing radiation and various cytostatic drugs in radiochemotherapy. DSBs do not only harm the integrity of the genome, but also promote the formation of mutations6,7. Therefore, different DSB repair pathways, and mechanisms to eliminate irreparably damaged cells like apoptosis have developed during evolution. The entire DNA damage response (DDR) is regulated by a complex network of signaling pathways that reach from DNA damage recognition and cell cycle arrest to allow for DNA repair, to programmed cell death or inactivation in case of repair failure8.
The presented flow cytometric method has been developed to investigate the DDR after genotoxic stress in one comprehensive assay that covers DSB induction and repair, as well as consequences of repair failure. It combines the measurement of the widely applied DSB marker &#947;H2AX with analysis of the cell cycle and induction of apoptosis, using classical subG1 analysis and more specific evaluation of caspase-3 activation.
The combination of these endpoints in one assay not only reduces time, labor and cost expenses, but also enables cell cycle-specific measurement of DSB induction and repair, as well as caspase-3 activation. Such analyses would not be possible with independently conducted assays, but they are highly relevant for a comprehensive understanding of the DNA damage response after genotoxic stress. Many anti-cancer drugs, such as cytostatic compounds, are directed against dividing cells and their efficiency is strongly dependent on the cell cycle stage. The availability of different DSB repair processes is also dependent on the cell cycle stage and pathway choice which is critical for the repair accuracy, and in turn determines the fate of the cell9,10,11,12. In addition, cell cycle-specific measurement of DSB levels is more accurate than pooled analysis, because DSB levels are not only dependent on the dose of a genotoxic compound or radiation, but also on the DNA content of the cell.
The method has been used to compare the efficacy of different radiotherapies to overcome resistance mechanisms in glioblastoma13 and to dissect the interplay between ionizing radiation and targeted drugs in osteosarcoma14,15 and atypical teratoid rhabdoid cancers16. Additionally, the described method has been widely used to analyze side effects of radio- and chemotherapy on mesenchymal stem cells17,18,19,20,21,22,23,24, which are essential for the repair of treatment-induced normal tissue damage and have a potential application in regenerative medicine.

<table border="0" cellpadding="0" cellspacing="0" style="width:1064px;" width="1063">
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			<td height="40" style="height:40px;width:405px;">1000 &#181;L filter tips</td>
			<td style="width:151px;">Nerbe plus</td>
			<td style="width:169px;">07-693-8300</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="96">
			<td height="96" style="height:97px;">100-1000 &#181;L pipette</td>
			<td>Eppendorf</td>
			<td>3123000063</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">12 x 75 mm Tubes with Cell Strainer Cap, 35 &#181;m mesh pore size</td>
			<td>BD Falcon</td>
			<td>352235</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">15 mL tubes</td>
			<td>BD Falcon</td>
			<td>352096</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">200 &#181;L filter tips</td>
			<td>Nerbe plus</td>
			<td>07-662-8300</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">20-200 &#181;L pipette</td>
			<td>Eppendorf</td>
			<td>3123000055</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">4&#8217;,6-Diamidin-2-phenylindol (DAPI)</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td style="width:339px;">Dissolve in water at 200 &#181;g/ml and store aliquots at -20 &#176;C</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:405px;">Alexa Fluor 488 anti-H2A.X Phospho (Ser139) Antibody, RRID: AB_2248011</td>
			<td>BioLegend</td>
			<td>613406</td>
			<td style="width:339px;">Dilute 1:20</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:405px;">Alexa Fluor 647 Rabbit Anti-Active Caspase-3 Antibody, AB_1727414</td>
			<td>BD Pharmingen</td>
			<td>560626</td>
			<td style="width:339px;">Dilute 1:20</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">BD FACSClean solution</td>
			<td>BD Biosciences</td>
			<td>340345</td>
			<td style="width:339px;">For cytometer cleaning routine after measurement</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">BD FACSRinse solution</td>
			<td>BD Biosciences</td>
			<td>340346</td>
			<td style="width:339px;">For cytometer cleaning routine after measurement</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dulbecco&#8217;s Phosphate Buffered Saline (PBS)</td>
			<td>Biochrom</td>
			<td>L 182</td>
			<td style="width:339px;">Dissolve in water to 1x concentration</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dulbecco&#39;s Modified Eagle&#39;s Medium with stable glutamin</td>
			<td>Biochrom</td>
			<td>FG 0415</td>
			<td style="width:339px;">Routine cell culture material for the example cell line used in the protocol</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ethanol absolute</td>
			<td>VWR</td>
			<td>20821.330</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Excel software</td>
			<td>Microsoft</td>
			<td></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">FBS Superior (fetal bovine serum)</td>
			<td>Biochrom</td>
			<td>S 0615</td>
			<td style="width:339px;">Routine cell culture material for the example cell line used in the protocol</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">FlowJo v10 software</td>
			<td>LLC</td>
			<td>online order</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fluoromount-G</td>
			<td>SouthernBiotech</td>
			<td>0100-01</td>
			<td style="width:339px;">Embedding medium for optional preparation of microscopic slides from stained samples</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">folded cellulose filters, grade 3hw</td>
			<td>NeoLab</td>
			<td>11416</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">LSRII or LSRFortessa cytometer</td>
			<td>BD Biosciences</td>
			<td></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">MG132</td>
			<td>Calbiochem</td>
			<td>474787</td>
			<td style="width:339px;">optional drug for apoptosis positive control</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Multifuge 3SR+</td>
			<td>Heraeus</td>
			<td></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Paraformaldehyde</td>
			<td>AppliChem</td>
			<td>A3813</td>
			<td style="width:339px;">Prepare 4.5% solution fresh. Dilute in PBS by heating to 80 &#176;C with slow stirring under the fume hood. Cover the flask with aluminium foil to prevent heat loss. Let the solution cool to room temperature and adjust the final volume. Pass the solution through a cellulose filter.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:405px;">Phospho-Histone H3 (Ser10) (D2C8) XP Rabbit mAb (Alexa Fluor&#174; 555 Conjugate) RRID: AB_10694639</td>
			<td>Cell Signaling Technology</td>
			<td>#3475</td>
			<td style="width:339px;">Dilute 3:200</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PIPETBOY acu 2</td>
			<td>Integra Biosciences</td>
			<td>155 016</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Serological pipettes, 10 mL</td>
			<td>Corning</td>
			<td>4488</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Serological pipettes, 25 mL</td>
			<td>Corning</td>
			<td>4489</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Serological pipettes, 5 mL</td>
			<td>Corning</td>
			<td>4487</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">SuperKillerTRAIL (modified TNF-related apoptosis-inducing ligand)</td>
			<td>Biomol</td>
			<td>AG-40T-0002-C020</td>
			<td style="width:339px;">optional drug for apoptosis positive control</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">T25 cell culture flasks</td>
			<td>Greiner bio-one</td>
			<td>690160</td>
			<td style="width:339px;">Routine cell culture material for the example cell line used in the protocol</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Trypsin/EDTA</td>
			<td>PAN Biotech</td>
			<td>P10-025500</td>
			<td style="width:339px;">Routine cell culture material for the example cell line used in the protocol</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">U87 MG glioblastoma cells</td>
			<td>ATCC</td>
			<td>ATCC-HTB-14</td>
			<td style="width:339px;">Example cell line used in the protocol</td>
		</tr>
	</tbody>
</table>

cell cycle-specific measurement of &#947;h2ax and apoptosis after genotoxic stress by flow cytometry

The presented method or slightly modified versions have been devised to study specific treatment responses and side effects of various anti-cancer treatments as used in clinical oncology. It enables a quantitative and longitudinal analysis of the DNA damage response after genotoxic stress, as induced by radiotherapy and a multitude of anti-cancer drugs. The method covers all stages of the DNA damage response, providing endpoints for induction and repair of DNA double-strand breaks (DSBs), cell cycle arrest and cell death by apoptosis in case of repair failure. Combining these measurements provides information about cell cycle-dependent treatment effects and thus allows an in-depth study of the interplay between cellular proliferation and coping mechanisms against DNA damage. As the effect of many cancer therapeutics including chemotherapeutic agents and ionizing radiation is limited to or strongly varies according to specific cell cycle phases, correlative analyses rely on a robust and feasible method to assess the treatment effects on the DNA in a cell cycle-specific manner. This is not possible with single-endpoint assays and an important advantage of the presented method. The method is not restricted to any particular cell line and has been thoroughly tested in a multitude of tumor and normal tissue cell lines. It can be widely applied as a comprehensive genotoxicity assay in many fields of oncology besides radio-oncology, including environmental risk factor assessment, drug screening and evaluation of genetic instability in tumor cells.

Human U87 or LN229 glioblastoma cells were irradiated with 4 Gy of photon or carbon ion radiation. Cell cycle-specific &#947;H2AX levels and apoptosis were measured at different time points up to 48 h after irradiation using the flow cytometric method presented here (Figure 3). In both cell lines, carbon ions induced higher &#947;H2AX peak levels that declined slower and remained significantly elevated at 24 to 48 h compared to photon radiation at the same physical dose (<strong class="xfig"...

Watch this Scientific Journal Video about Cell Cycle-specific Measurement of ÃŽÂ³H2AX and Apoptosis After Genotoxic Stress by Flow Cytometry at JoVE.com

Cell Cycle-specific Measurement of ÃƒÅ½Ã‚Â³H2AX and Apoptosis After Genotoxic Stress by Flow Cytometry

The presented method combines the quantitative analysis of DNA double-strand breaks (DSBs), cell cycle distribution and apoptosis to enable cell cycle-specific evaluation of DSB induction and repair as well as the consequences of repair failure.

Cell Cycle-specific Measurement of &#947;H2AX and Apoptosis After Genotoxic Stress by Flow Cytometry

The presented method or slightly modified versions have been devised to study specific treatment responses and side effects of various anti-cancer ...

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