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 Î³H2AX 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 Î³H2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks at JoVE.com

Immunofluorescence Microscopy of &amp;#947;H2AX 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 ...

The overall goal of gamma-H2AX and 53BP1 immunofluorescence microscopy is to analyze the formation and repair of DNA double-strand breaks in various tissues. This method can help answer key questions in cancer research, such as how genetic instability arises in tumor cells. The main advantage of this technique is that single DNA double-string breaks can be visualized, and that the repair processes can be analyzed.Demonstrating the procedure will be Susanne Brendel, a technician of our laboratory. Begin the experiment by preparing solutions, starting with the anti-coagulant stock solution with 200 International Units of heparin per milliliter in 0.9 per cent sodium chloride. Fill each of the collection tubes with two milliliters of anticoagulant stock solution before withdrawal of the blood or bone marrow samples.Next, prepare the 10X Lysis solution for red cells with 82.91 grams ammonium chloride, 7.91 grams ammonium bicarbonate, and two milliliters of 0.5 molar ethylenediamenetetracedic acid solution to a pH of 8.0 by dissolving the agents in double distilled water for a total volume of one liter. Then, prepare the fixation solution by adding 8.3 microliters of a one molar potassium hydroxide solution to 360 milligrams of paraformaldehyde, or PFA, in a microtiter tube. Fill the tube with phosphate buffered saline, or PBS, to the one milliliter mark of the tube and heat the tube in a heating block at 95 degrees Celsius to obtain one milliliter of a 36 per cent PFA solution.Transfer the one milliliter of 36 per cent PFA solution to a 15 milliliter tube. And add eight milliliters of PBS to obtain a nine milliliter four per cent PFA stock solution. Aliquot the four per cent PFA stock solution and store it at negative 18 degrees Celsius until use for cell fixation.Next, prepare the permeabilization solution by adding 50 microliters of Octoxynol 9 to 50 milliliters of PBS to obtain a solution of approximately 0.1 per cent Octoxynol 9. Finally, prepare five per cent and two per cent blocking solutions by mixing the protein blocking agent with PBS. To prepare the blood or bone marrow samples, retrieve the tubes filled with two milliliters of the anticoagulant stock solution.Withdraw seven milliliters of blood or bone marrow per tube under sterile conditions by venepuncture or bone marrow puncture respectively. Store the samples over night at room temperature. After storage overnight, dilute the heparinized blood sample one to one with PBS and the heparinized bone marrow sample in a ratio of one to three with PBS.Suspend the cells gently by drawing them in and out of the pipette two times. Next, add one volume of density gradient medium to a fresh tube. Gently layer the diluted blood or bone marrow on top of the density gradient medium and take care not to mix the two layers.Centrifuge the tube for 30 minutes at room temperature and 400 G.Draw the upper plasma layer off with a pipette. Then, carefully harvest the mononuclear cells in the layer above the density gradient medium. Transfer the mononuclear cells into a fresh tube and add at least three volumes of PBS.Suspend the cells gently by drawing them in and out of the pipette two times. Centrifuge the tube. After the spin, remove the supernatant.Add 10 milliliters of four degrees Celsius 1x Lysis solution for red cells. Re-suspend the cells gently by drawing them in and out of the pipette two times and place the tube on ice for five minutes. Then, add 30 milliliters of PBS at four degrees Celsius and centrifuge the tube.Use CD34 micro beads and cell separation columns for the isolation of CD34 positive cells. Prepare two cyto-spins with mononuclear cells of the patient samples by centrifugation of one times 10 the five cells for each preparation. Fix the cells with 200 microliters of four per cent PFA for 10 minutes.After fixation, wash the cells gently three times with 30 milliliters of PBS for five minutes each on a shaker. Then, permeablize the cells with 200 microliters of 0.1 per cent Octoxynol 9 for 10 minutes. Wash the cells gently three times with 30 milliliters of five per cent blocking solution for five minutes each on a lab shaker.Block the cells in 30 milliliters of fresh five per cent blocking solution for one hour. The fixation and permeablization of the cells with four per cent paraformaldehyde in 0.1 per cent Octoxynol 9 preserves the gamma-H2AX and 53BP1 foci distinctly. Incubate one preparation of the cells with a mouse monoclonal anti-gamma-H2AX antibody and the other preparation of the cells with a mouse monoclonal anti-gamma-H2AX antibody and a polyclonal rabbit anti-53BP1 antibody overnight at four degrees Celsius.The use of proven anti-gamma-H2AX and anti-53BP1 antibodies is highly recommended. After incubation, wash the cells gently three times with 30 milliliters of two per cent blocking solution for each five minutes on a lab shaker. Next, incubate the first preparation of the cells with an Alexa 488 conjugated goat anti-mouse secondary antibody diluted one in 500 in two per cent blocking solution.Incubate the second preparation of the cells with an Alexa 488 conjugated goat anti-mouse secondary antibody and an Alexa 555 conjugated donkey anti-rabbit secondary antibody diluted one in 500 into two per cent blocking solution. Wash the cells gently three times with 30 milliliters of PBS on a laboratory shaker. Remove the PBS and mount the cells with mounting medium.Cautiously put a cover slip on top of the mounting medium so that no air bubbles are embedded. Wait at least three hours for the mounting medium to harden before analyzing the cells by fluorescence microscopy. Finally, analyze the gamma-H2AX and 53BP1 foci in the cell nuclei with a fluorescence microscope equipped with filters for DAPI, Alexa 488, and Cy3 during imaging at a 100x objected magnification.Analysis of gamma-H2AX foci in cells is most accurate in the G0 G1 phase and the G2 phase when gamma-H2AX foci appear as distinct fluorescent dots as shown in this representative image of fibroblasts. In contrast, analysis of gamma-H2AX foci in cells during the S phase is complicated by dispersed pan-nuclear gamma-H2AX speckles caused by the replication process. Immunofluorescence staining of gamma-H2AX was used for the quantification of DNA double-strand breaks in irradiated tissues and elucidated that the levels of gamma-H2AX foci in irradiated lymphocytes are proportional to the irradiation dose.Co-localization of gamma-H2AX and 53BP1 foci in CD34 positive cells of a patient with acute myeloid leukemia indicates promotion of 53BP1 mediated nonhomologous end-joining repair mechanisms at sites of DNA double strand breaks. After this watching this video, you should have a good understanding of how to perform gamma-H2AX and 53BP1 immunofluorescence microscopy for the analysis of DNA double strand breaks. Following this procedure, other methods like immunoblotting of phosphoryrelated ATM, ATR, checkpoint kinase one, checkpoint kinase two and TP53 can be performed in tumor cells in order to analyze alterations of the DNA damage response.

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Research

JoVE Journal

Cancer Research

Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular biology techniques have clarified structure, enzymatic functions, and kinetics of repair proteins, there is still a need to understand how repair is coordinated within the nucleus. Laser micro-irradiation offers a powerful tool for inducing DNA damage and monitoring the recruitment of repair proteins. Induction of DNA damage by laser micro-irradiation can occur with a range of wavelengths, and users can reliably induce single strand breaks, base lesions and double strand breaks with a range of doses. Here, laser micro-irradiation is used to examine repair of single and double strand breaks induced by two common confocal laser wavelengths, 355 nm and 405 nm. Further, proper characterization of the applied laser dose for inducing specific damage mixtures is described, so users can reproducibly perform laser micro-irradiation data acquisition and analysis.

Confocal fluorescence microscopy and laser micro-irradiation offer tools for inducing DNA damage and monitoring the response of DNA repair proteins in selected sub-nuclear areas. This technique has significantly advanced our knowledge of damage detection, signaling, and recruitment. This manuscript demonstrates these technologies to examine single and double strand break repair.

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular ...

Characterization of Tumor Cells Using a Medical Wire for Capturing Circulating Tumor Cells: A 3D Approach Based on Immunofluorescence and DNA FISH

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a better understanding of tumor heterogeneity and thus facilitate the selection of patients for personalized treatment. However, this is hampered because of the rarity of CTCs. We present an innovative approach for sampling a high volume of the patient blood and obtaining information about presence, phenotype, and gene translocation of CTCs. The method combines immunofluorescence staining and DNA fluorescent-in-situ-hybridization (DNA FISH) and is based on a functionalized medical wire. This wire is an innovative device that permits the in vivo isolation of CTCs from a large volume of peripheral blood. The blood volume screened by a 30-min administration of the wire is approximately 1.5-3 L. To demonstrate the feasibility of this approach, epithelial cell adhesion molecule (EpCAM) expression and the chromosomal translocation of the ALK gene were determined in non-small-cell lung cancer (NSCLC) cell lines captured by the functionalized wire and stained with an immuno-DNA FISH approach. Our main challenge was to perform the assay on a 3D structure, the functionalized wire, and to determine immuno-phenotype and FISH signals on this support using a conventional fluorescence microscope. The results obtained indicate that catching CTCs and analyzing their phenotype and chromosomal rearrangement could potentially represent a new companion diagnostic approach and provide an innovative strategy for improving personalized cancer treatments.

We present a new approach to characterize tumor cells. We combined immunofluorescence with DNA fluorescent-in-situ-hybridization to evaluate cells captured by a functionalized medical wire capable of in vivo enriching CTCs directly from patient blood.

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a ...

Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair complexes. This process is regulated by a myriad of proteins that promote the association and disassociation of proteins to these lesions. Thanks in large part to the ability to perform functional screens of a vast library of proteins, there is a greater appreciation of the genes necessary for the double-strand DNA break repair. Often knockout or chemical inhibitor screens identify proteins involved in repair processes by using increased toxicity as a marker for a protein that is required for DSB repair. Although useful for identifying novel cellular proteins involved in maintaining genome fidelity, functional analysis requires the determination of whether the protein of interest promotes localization, formation, or resolution of repair complexes. 
The accumulation of repair proteins can be readily detected as distinct nuclear foci by immunofluorescence microscopy. Thus, association and disassociation of these proteins at sites of DNA damage can be accessed by observing these nuclear foci at representative intervals after the induction of double-strand DNA breaks. This approach can also identify mis-localized repair factor proteins, if repair defects do not simultaneously occur with incomplete delays in repair. In this scenario, long-lasting double-strand DNA breaks can be engineered by expressing a rare cutting endonuclease (e.g., I-SceI) in cells where the recognition site for the said enzyme has been integrated into the cellular genome. The resulting lesion is particularly hard to resolve as faithful repair will reintroduce the enzyme's recognition site, prompting another round of cleavage. As a result, differences in the kinetics of repair are eliminated. If repair complexes are not formed, localization has been impeded. This protocol describes the methodology necessary to identify changes in repair kinetics as well as repair protein localization.

Repair of double-strand DNA breaks is a dynamic process, requiring not only formation of repair complexes at the breaks, but also their resolution after the lesion is addressed. Here, we use immunofluorescence microscopy for transient and long-lasting double-stranded breaks as a tool to dissect this genome maintenance mechanism.

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair ...

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in&#160;patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.

Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular ...

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 response induced by neutron-gamma mixed-beam used in boron neutron capture therapy (BNCT). Specifically, the proposed methodology is applied for the detection of repair proteins activation which can be visualized as foci using antibodies specific to DNA double-strand breaks (DNA-DSBs). DNA repair foci were assessed by immunofluorescence in colon cancer cells (HCT-116) after irradiation with the neutron-mixed beam. DNA-DSBs are the most genotoxic lesions and are repaired in mammalian cells by two major pathways: non-homologous end-joining pathway (NHEJ) and homologous recombination repair (HRR). The frequencies of foci, immunochemically stained, for commonly used markers in radiobiology like &#947;-H2AX, 53BP1 are associated with DNA-DSB number and are considered as efficient and sensitive markers for monitoring the induction and repair of DNA-DSBs. It was established that &#947;-H2AX foci attract repair proteins, leading to a higher concentration of repair factors near a DSB. To monitor DNA damage at the cellular level, immunofluorescence analysis for the presence of DNA-PKcs representative repair protein foci from the NHEJ pathway and Rad52 from the HRR pathway was planned. We have developed and introduced a reliable immunofluorescence staining protocol for the detection of radiation-induced DNA damage response with antibodies specific for repair factors from NHEJ and HRR pathways and observed radiation-induced foci (RIF). The proposed methodology can be used for investigating repair protein that is highly activated in the case of neutron-mixed beam radiation, thereby indicating the dominance of the repair pathway.

Radiation-induced DNA damage response activated by neutron mixed-beam used in boron neutron capture therapy (BNCT) has not been fully established. This protocol provides a step-by-step procedure to detect radiation-induced foci (RIF) of repair proteins by immunofluorescence staining in human colon cancer cell lines after irradiation with the neutron-mixed beam.

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 as well as for the treatment selection and its response in cancer patients. The current gold standard method for detecting tumor mutations involves a genetic test of tumor DNA by means of tumor biopsies. However, this invasive method is difficult to be performed repeatedly as a follow-up test of the tumor mutational repertoire. Liquid biopsy is a new and emerging technique for detecting tumor mutations as an easy-to-use and non-invasive biopsy approach.
Cancer cells multiply rapidly. In parallel, numerous cancer cells undergo apoptosis. Debris from these cells are released into a patient&#8217;s circulatory system, together with finely fragmented DNA pieces, called cell-free DNA (cfDNA) fragments, which carry tumor DNA mutations. Therefore, for identifying cfDNA based biomarkers using liquid biopsy technique, blood samples are collected from the cancer patients, followed by the separation of plasma and buffy coat. Next, plasma is processed for the isolation of cfDNA, and the respective buffy coat is processed for the isolation of a patient&#39;s genomic DNA. Both nucleic acid samples are then checked for their quantity and quality; and analyzed for mutations using next-generation sequencing (NGS) techniques.
In this manuscript, we present a detailed protocol for liquid biopsy, including blood collection, plasma, and buffy coat separation, cfDNA and germline DNA extraction, quantification of cfDNA or germline DNA, and cfDNA fragment enrichment analysis.

In this paper we present a detailed protocol for non-invasive liquid biopsy technique, including blood collection, plasma and buffy coat separation, cfDNA and germline DNA extraction, quantification of cfDNA or germline DNA, and cfDNA fragment enrichment analysis.

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 animal models have been generated, including the widely used orthotopic mouse models, where cancer cells are injected into the mammary fat pad. However, these models cannot help monitor tumor growth kinetics and metastatic colonization. Cutting-edge tools to monitor cancer cells in real time in mice will significantly advance the understanding of tumor biology. 
Here, breast cancer cell lines stably expressing luciferase and green fluorescent protein (GFP) were established. Specifically, this technique contains two sequential steps initiated by measuring the luciferase activity in vitro and followed by the implantation of the cancer cells into mammary fat pads of nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice. After the injection, both the tumor growth and metastatic colonization are monitored in real time by the noninvasive bioluminescence imaging system. Then, the quantification of GFP-expressing metastases in the lungs will be examined by fluorescence microscopy to validate the observed bioluminescence results. This sophisticated system combining luciferase and fluorescence-based detection tools evaluates cancer metastasis in vivo, which has great potential for use in breast cancer therapeutics and disease management.

Here, we describe a noninvasive monitoring method involving luciferase and green fluorescent protein expression in various breast cancer cell lines. This protocol provides a technique to monitor tumor formation and metastatic colonization in real time in mice.

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

Immunofluorescence is one of the most widely used techniques to visualize target antigens with high sensitivity and specificity, allowing for the accurate identification and localization of proteins, glycans, and small molecules. While this technique is well-established in two-dimensional (2D) cell culture, less is known about its use in three-dimensional (3D) cell models. Ovarian cancer organoids are 3D tumor models that recapitulate tumor cell clonal heterogeneity, the tumor microenvironment, and cell-cell and cell-matrix interactions. Thus, they are superior to cell lines for the evaluation of drug sensitivity and functional biomarkers. Therefore, the ability to utilize immunofluorescence on primary ovarian cancer organoids is extremely beneficial in understanding the biology of this cancer. The current study describes the technique of immunofluorescence to detect DNA damage repair proteins in high-grade serous patient-derived ovarian cancer organoids (PDOs). After exposing the PDOs to ionizing radiation, immunofluorescence is performed on intact organoids to evaluate nuclear proteins as foci. Images are collected using z-stack imaging on confocal microscopy and analyzed using automated foci counting software. The described methods allow for the analysis of temporal and special recruitment of DNA damage repair proteins and colocalization of these proteins with cell-cycle markers.

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

The present protocol describes methods for evaluating DNA damage repair proteins in patient-derived ovarian cancer organoids. Included here are comprehensive plating and staining methods, as well as detailed, objective quantification procedures.

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

The tumor microenvironment (TME) is composed of a plethora of different cell types, such as cytotoxic immune cells and immunomodulatory cells. Depending on its composition and the interactions between cancer cells and peri-tumoral cells, the TME may affect cancer progression. The characterization of tumors and their complex microenvironment could improve the understanding of cancer diseases and may help scientists and clinicians to discover new biomarkers.
We recently developed several multiplex immunofluorescence (mIF) panels based on tyramide signal amplification (TSA) for the characterization of the TME in colorectal cancer, head and neck squamous cell carcinoma, melanoma, and lung cancer. Once the staining and scanning of the corresponding panels are completed, the samples are analyzed on an image analysis software. The spatial position and the staining of each cell are then exported from this quantification software into R. We developed R scripts that allow us not only to analyze the density of each cell type in several tumor compartments (e.g. the center of the tumor, the margin of the tumor, and the stroma) but also to perform distance-based analyses between different cell types.
This particular workflow adds a spatial dimension to the classical density analysis already routinely performed for several markers. mIF analysis could allow scientists to have a better understanding of the complex interaction between cancer cells and the TME and to discover new predictive biomarkers of response to treatments, such as immune checkpoint inhibitors, and targeted therapies.

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

In this article, a protocol for manual tyramide signal amplification (TSA) multiplex immunofluorescence (mIF) combined with image analysis and spatial analysis is described. This protocol can be used with formalin-fixed paraffin-embedded (FFPE) sections for the staining of two to six antigens per slide depending on the slide scanner available in the laboratory.

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

Diagnosis of Vitreoretinal Lymphoma Using Cell-Free DNA

Cell-Free DNA Extraction of Vitreous and Aqueous Humor Specimens for Diagnosis and Monitoring of Vitreoretinal Lymphoma

Vitreoretinal lymphoma (VRL) represents an aggressive lymphoma, often categorized as primary central nervous system diffuse large B-cell lymphoma. To diagnose VRL, specimens such as vitreous humor and, more recently, aqueous humor are collected. Diagnostic testing for VRL on these specimens includes cytology, flow cytometry, and molecular testing. However, both cytopathology and flow cytometry, along with molecular testing using cellular DNA, necessitate intact whole cells. The challenge lies in the fact that vitreous and aqueous humor typically have low cellularity, and many cells get destroyed during collection, storage, and processing. Moreover, these specimens pose additional difficulties for molecular testing due to the high viscosity of vitreous humor and the low volume of both vitreous and aqueous humor. This study proposes a method for extracting cell-free DNA from vitreous and aqueous specimens. This approach complements the extraction of cellular DNA or allows the cellular component of these specimens to be utilized for other diagnostic methods, including cytology and flow cytometry.

Author Spotlight: Advancing VRL Diagnosis Using Cell-Free DNA Extraction from Vitreous Humor 

A procedure to extract cell-free DNA from vitreous and aqueous humor to perform molecular studies for diagnosing vitreoretinal lymphoma is established here. The method offers the ability to concurrently extract DNA from the cellular component of the sample or to reserve it for ancillary testing.

Vitreoretinal lymphoma (VRL) represents an aggressive lymphoma, often categorized as primary central nervous system diffuse large B-cell lymphoma. To ...