Name: immunofluorescence microscopy of &#947;h2ax and 53bp1 for analyzing the formation and repair of dna double-strand breaks
Uploaded: 2017-11-03T14:00:00
Description: Watch this Scientific Journal Video about Immunofluorescence Microscopy of ÃŽÂ³H2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks at JoVE.com

The project was supported by the German Jos&#233; Carreras Leukemia Foundation (DJCLS 14 R/2017).

All methods described here have been approved by the Ethics Committee II of the Medical Faculty Mannheim of the Heidelberg University. Written informed consent was obtained from all individuals.
1. Preparation of Materials
<ol>
	<li>Anticoagulant stock solution: Prepare an anticoagulant stock solution of 200 I.U. heparin per mL in 0.9% sodium chloride. Fill each of the collection tubes (draw volume 9 mL) with 2 mL of the anticoagulant stock solution before withdrawal of the ...

<ol>
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K., 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=Evidence+for+ongoing+DNA+damage+in+multiple+myeloma+cells+as+revealed+by+constitutive+phosphorylation+of+H2AX.">Evidence for ongoing DNA damage in multiple myeloma cells as revealed by constitutive phosphorylation of H2AX.</a> Leukemia. 25 (8), 1344-1353 (2011).</li><li>Yu, T., MacPhail, S. H., Banath, J. P., Klokov, D., Olive, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endogenous+expression+of+phosphorylated+histone+H2AX+in+tumors+in+relation+to+DNA+double-strand+breaks+and+genomic+instability.">Endogenous expression of phosphorylated histone H2AX in tumors in relation to DNA double-strand breaks and genomic instability.</a> DNA Repair (Amst). 5 (8), 935-946 (2006).</li><li>Sedelnikova, O. A., 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=GammaH2AX+in+cancer+cells:+a+potential+biomarker+for+cancer+diagnostics,+prediction+and+recurrence.">GammaH2AX in cancer cells: a potential biomarker for cancer diagnostics, prediction and recurrence.</a> Cell Cycle. 5 (24), 2909-2913 (2006).</li><li>Chen, E., 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=JAK2V617F+promotes+replication+fork+stalling+with+disease-restricted+impairment+of+the+intra-S+checkpoint+response.">JAK2V617F promotes replication fork stalling with disease-restricted impairment of the intra-S checkpoint response.</a> Proc Natl Acad Sci U S A. 111 (42), 15190-15195 (2014).</li><li>Popp, H. D., 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=Increase+of+DNA+damage+and+alteration+of+the+DNA+damage+response+in+myelodysplastic+syndromes+and+acute+myeloid+leukemias.">Increase of DNA damage and alteration of the DNA damage response in myelodysplastic syndromes and acute myeloid leukemias.</a> Leuk Res. 57, 112-118 (2017).</li><li>Panier, S., Boulton, 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=Double-strand+break+repair:+53BP1+comes+into+focus.">Double-strand break repair: 53BP1 comes into focus.</a> Nat Rev Mol Cell Biol. 15 (1), 7-18 (2014).</li><li>Escribano-Diaz, 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> Mol Cell. 49 (5), 872-883 (2013).</li><li>Boyum, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+White+Blood+Cells.">Separation of White Blood Cells.</a> Nature. 204, 793-794 (1964).</li><li>Boyum, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+mononuclear+cells+and+granulocytes+from+human+blood.+Isolation+of+monuclear+cells+by+one+centrifugation,+and+of+granulocytes+by+combining+centrifugation+and+sedimentation+at+1+g.">Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g.</a> Scand J Clin Lab Invest Suppl. 97, 77-89 (1968).</li><li>Bain, B., Pshyk, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+reactivity+in+mixed+leukocyte+cultures+after+separation+of+mononuclear+cells+on+Ficoll-Hypaque.">Enhanced reactivity in mixed leukocyte cultures after separation of mononuclear cells on Ficoll-Hypaque.</a> Transplant Proc. 4 (2), 163-164 (1972).</li><li>Popp, H. D., 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=Leukocyte+DNA+damage+after+reduced+and+conventional+absorbed+radiation+doses+using+3rd+generation+dual-source+CT+technology.">Leukocyte DNA damage after reduced and conventional absorbed radiation doses using 3rd generation dual-source CT technology.</a> Eur J Radiol Open. 3, 134-137 (2016).</li><li>Rothkamm, K., Balroop, S., Shekhdar, J., Fernie, P., Goh, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+DNA+damage+after+multi-detector+row+CT:+a+quantitative+biomarker+of+low-level+radiation+exposure.">Leukocyte DNA damage after multi-detector row CT: a quantitative biomarker of low-level radiation exposure.</a> Radiology. 242 (1), 244-251 (2007).</li><li>Lobrich, 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+formation+and+repair+of+DNA+double-strand+breaks+after+computed+tomography+examinations.">In vivo formation and repair of DNA double-strand breaks after computed tomography examinations.</a> Proc Natl Acad Sci U S A. 102 (25), 8984-8989 (2005).</li><li>Jamur, M. C., Oliver, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+fixatives+for+immunostaining.">Cell fixatives for immunostaining.</a> Methods Mol Biol. 588, 55-61 (2010).</li><li>Jamur, M. C., Oliver, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeabilization+of+cell+membranes.">Permeabilization of cell membranes.</a> Methods Mol Biol. 588, 63-66 (2010).</li><li>Rothkamm, K., Lobrich, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+a+lack+of+DNA+double-strand+break+repair+in+human+cells+exposed+to+very+low+x-ray+doses.">Evidence for a lack of DNA double-strand break repair in human cells exposed to very low x-ray doses.</a> Proc Natl Acad Sci U S A. 100 (9), 5057-5062 (2003).</li></ol>

Immunofluorescence microscopy of &#947;H2AX and 53BP1 is a useful method for analyzing formation and repair of DSB in a broad spectrum of research areas. Critical parameters that influence the outcome of the experiments are the phase of the cell cycle, the agents used for the fixation and permeabilization of the cells, the choice of the antibodies, and the hardware and software of the fluorescence microscope.
The influence of the cell cycle on &#947;H2AX foci patterns was demonstrated by expon...

DNA is continuously damaged by endogenous (e.g., replication stress, reactive oxygen species, intrinsic instability of DNA) and exogenous (e.g., chemical radicals, irradiation) sources (Figure 1)1,2,3,4. Among DNA damage, DNA double-strand breaks (DSB) are particularly serious lesions and may induce cell death or carcinogenesis. About 50 DSB may arise per cell and cell cycle5. In mammalian cells, homologous recombination (HR) and nonhomologous end-joining (NHEJ) developed as major pathways for the repair of DSB (Figure 2). HR occurs in late S/G2 phase and uses an intact sister chromatid as a template for potentially error-free repair. In comparison, NHEJ is active throughout the cell cycle and potentially mutagenic as base pairs may be added or resected before ligation of the broken ends. In addition, alternative end-joining may be engaged as a slow mutagenic back-up repair mechanism in case of NHEJ deficiency6,7.
DSB induce the phosphorylation of the histone H2AX at Serine 139 in a region of several megabase pairs around each DSB. The forming nuclear foci are named &#947;H2AX foci and are detectable by immunofluorescence microscopy8. &#947;H2AX promotes the recruitment of further DSB-responsive proteins and is involved in chromatin remodeling, DNA repair, and signal transduction9. As each &#947;H2AX focus is considered to represent a single DSB, the quantification of DSB by immunofluorescence microscopy is possible, which has been demonstrated in cancer cell lines and patient specimens10,11,12,13,14. 53BP1 (p53 binding protein 1) is another key protein in mediating DSB repair. It is involved in the recruitment of DSB-responsive proteins, checkpoint signaling, and the synapsis of DSB ends15. In addition, 53BP1 plays a critical role in the DSB repair pathway choice. It triggers the repair of DSB towards NHEJ while HR is prevented16. Considering the genuine functions of &#947;H2AX and 53BP1 in DSB repair, the simultaneous analysis of &#947;H2AX and 53BP1 by immunofluorescence microscopy may be a useful method for the precise analysis of the formation and repair of DSB.
This manuscript provides a step-by-step protocol for performing immunofluorescence microscopy of &#947;H2AX and 53BP1 in cell nuclei. Specifically, the technique is applied in normal fibroblasts of the cell line NHDF, in x-ray irradiated lymphocytes of a healthy individual and in CD34+ cells of a patient with acute myeloid leukemia. The details of the method are pointed out in the context of the presented results.

<table border="0" cellpadding="0" cellspacing="0" width="630">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">RPMI medium</td>
			<td style="width:119px;">Sigma-Aldrich</td>
			<td style="width:104px;">R0883</td>
			<td style="width:200px;">Medium for cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Heparin sodium</td>
			<td style="width:119px;">ratiopharm</td>
			<td style="width:104px;">PZN 3029843</td>
			<td style="width:200px;">&#160;Heparin 5,000 I.U. / mL</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Sodium chloride solution 0.9%</td>
			<td style="width:119px;">B. Braun</td>
			<td style="width:104px;">PZN 1957154</td>
			<td style="width:200px;">Component of the anticoagulant stock solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Ficoll-Paque Premium</td>
			<td style="width:119px;">GE Healthcare</td>
			<td style="width:104px;">17-5442-02</td>
			<td style="width:200px;">Medium for isolation of mononuclear cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Trypsin solution 10X</td>
			<td style="width:119px;">Sigma-Aldrich</td>
			<td style="width:104px;">59427C</td>
			<td style="width:200px;">Enzyme for dissociation of fibroblasts in cell culture</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">CD34 MicroBead Kit</td>
			<td style="width:119px;">Miltenyi Biotec</td>
			<td style="width:104px;">130-046-702</td>
			<td style="width:200px;">Isolation of CD34+ myeloid progenitor cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Diagnostic microscope slides</td>
			<td>Thermo Scientific</td>
			<td>ER-203B-CE24</td>
			<td style="width:200px;">Microscope slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Megafuge 1.0 R</td>
			<td style="width:119px;">Heraeus</td>
			<td style="width:104px;">75003060</td>
			<td style="width:200px;">&#160;Tabletop centrifuge&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Cytospin device</td>
			<td style="width:119px;"></td>
			<td style="width:104px;"></td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Lid</td>
			<td style="width:119px;">Heraeus</td>
			<td>76003422</td>
			<td style="width:200px;">Lid for working without micro-tubes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Cyto container</td>
			<td style="width:119px;">Heraeus</td>
			<td style="width:104px;">75003416</td>
			<td style="width:200px;">Cyto container with 2 conical bores</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Clip carrier</td>
			<td style="width:119px;">Heraeus</td>
			<td style="width:104px;">75003414</td>
			<td style="width:200px;">Carrier for holding a cyto container and a slide</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Support insert</td>
			<td style="width:119px;">Heraeus</td>
			<td style="width:104px;">75003417</td>
			<td style="width:200px;">Support insert for holding a clip carrier</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Triton X-100 (Octoxinol 9)</td>
			<td style="width:119px;">Thermo Scientific</td>
			<td style="width:104px;">85112</td>
			<td style="width:200px;">Detergent for permeabilization of cell membranes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Potassium hydroxide solution 1M</td>
			<td style="width:119px;">Merck Millipore</td>
			<td style="width:104px;">109107</td>
			<td style="width:200px;">Necessary for preparing the paraformaldehyde solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Paraformaldehyde</td>
			<td style="width:119px;">Sigma-Aldrich</td>
			<td style="width:104px;">P6148</td>
			<td style="width:200px;">Fixation agent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Phosphate buffered saline</td>
			<td style="width:119px;">Sigma-Aldrich</td>
			<td style="width:104px;">D8537</td>
			<td style="width:200px;">Balanced salt solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Chemiblocker</td>
			<td style="width:119px;">Merck Millipore</td>
			<td style="width:104px;">2170</td>
			<td style="width:200px;">Blocking agent</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Mouse monoclonal anti-&#947;H2AX antibody (JBW301)</td>
			<td style="width:119px;">Merck Millipore</td>
			<td style="width:104px;">05-636</td>
			<td style="width:200px;">Primary antibody for detection of &#947;H2AX</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Polyclonal rabbit anti-53BP1 antibody (NB100-304)</td>
			<td style="width:119px;">Novus Biologicals</td>
			<td style="width:104px;">NB100-304</td>
			<td style="width:200px;">Primary antibody for detection of 53BP1</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Alexa Fluor 488-conjugated goat anti-mouse antibody</td>
			<td style="width:119px;">Invitrogen</td>
			<td style="width:104px;">A-11001</td>
			<td style="width:200px;">Secondary antibody</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Alexa Fluor 555-conjugated goat anti-rabbit antibody</td>
			<td style="width:119px;">Invitrogen</td>
			<td style="width:104px;">A-21428</td>
			<td style="width:200px;">Secondary antibody</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Vectashield mounting medium</td>
			<td style="width:119px;">Vector Laboratories</td>
			<td style="width:104px;">H-1200</td>
			<td style="width:200px;">Contains DAPI for staining of DNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Axio Scope.A1</td>
			<td style="width:119px;">Zeiss</td>
			<td style="width:104px;">490035</td>
			<td style="width:200px;">Fluorescence microscope</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Cool Cube 1 CCD camera</td>
			<td style="width:119px;">Metasystems</td>
			<td style="width:104px;">H-0310-010-MS</td>
			<td style="width:200px;">Camera system for digital recording</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Isis software</td>
			<td style="width:119px;">Metasystems</td>
			<td style="width:104px;">Not applicable</td>
			<td style="width:200px;">Microscope software</td>
		</tr>
	</tbody>
</table>

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

DNA double-strand breaks (DSB) are serious DNA lesions. Analysis of the formation and repair of DSB is relevant in a broad spectrum of research areas including genome integrity, genotoxicity, radiation biology, aging, cancer, and drug development. In response to DSB, the histone H2AX is phosphorylated at Serine 139 in a region of several megabase pairs forming discrete nuclear foci detectable by immunofluorescence microscopy. In addition, 53BP1 (p53 binding protein 1) is another important DSB-responsive protein promoting repair of DSB by nonhomologous end-joining while preventing homologous recombination. According to the specific functions of &#947;H2AX and 53BP1, the combined analysis of &#947;H2AX and 53BP1 by immunofluorescence microscopy may be a reasonable approach for a detailed analysis of DSB. This manuscript provides a step-by-step protocol supplemented with methodical notes for performing the technique. Specifically, the influence of the cell cycle on &#947;H2AX foci patterns is demonstrated in normal fibroblasts of the cell line NHDF. Further, the value of the &#947;H2AX foci as a biomarker is depicted in x-ray irradiated lymphocytes of a healthy individual. Finally, genetic instability is investigated in CD34+ cells of a patient with acute myeloid leukemia by immunofluorescence microscopy of &#947;H2AX and 53BP1.

Analysis of &#947;H2AX foci in cells is most accurate in the G0/G1 phase and the G2 phase when &#947;H2AX foci appear as distinct fluorescent dots (Figure 5A). In contrast, analysis of &#947;H2AX foci in cells during the S phase is complicated by dispersed pan-nuclear &#947;H2AX speckles caused by the replication process (Figure 5B).
Fixation of the cells was performed ...

Watch this Scientific Journal Video about Immunofluorescence Microscopy of ÃŽÂ³H2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks at JoVE.com

Immunofluorescence Microscopy of ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â³H2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks

This manuscript provides a protocol for the analysis of DNA double-strand breaks by immunofluorescence microscopy of &#947;H2AX and 53BP1.

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

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

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Monoclonal antibodies are high affinity multifunctional drugs that work by variable independent mechanisms to eliminate cancer cells. Over the last few ...

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

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

Detection of Cell-Free DNA in Blood Plasma Samples of Cancer Patients

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis ...

Monitoring Breast Cancer Growth and Metastatic Colony Formation in Mice using Bioluminescence

Breast cancer is a frequent heterogeneous malignancy and the second leading cause of mortality in women, mainly due to distant organ metastasis. Several ...

Visualizing DNA Damage Repair Proteins in Patient-Derived Ovarian Cancer Organoids via Immunofluorescence Assays

Visualizing DNA Damage Repair Proteins in Patient-Derived Ovarian Cancer Organoids via Immunofluorescence Assays

Immunofluorescence is one of the most widely used techniques to visualize target antigens with high sensitivity and specificity, allowing for the accurate ...

Multiplex Immunofluorescence and Spatial Image Analysis of the Tumor Microenvironment

Multiplex Immunofluorescence Combined with Spatial Image Analysis for the Clinical and Biological Assessment of the Tumor Microenvironment

Multiplex Immunofluorescence Combined with Spatial Image Analysis for the Clinical and Biological Assessment of the Tumor Microenvironment (Video) | JoVE 

The tumor microenvironment (TME) is composed of a plethora of different cell types, such as cytotoxic immune cells and immunomodulatory cells. Depending ...