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 blood or bone marrow samples.</li>
	<li>Lysis solution for red cells: Prepare the 10x&#160;lysis solution for red cells with 82.91 g ammonium chloride, 7.91 g ammonium bicarbonate, and 2 mL of 0.5 M ethylenediaminetetraacetic acid solution to a pH of 8.0 by dissolving the agents in double-distilled water for a total volume of 1 L.</li>
	<li>Fixation solution: Add 8.3 &#181;L of a 1 M potassium hydroxide solution to 360 mg paraformaldehyde (PFA) in a microtiter tube. Fill the tube with phosphate buffered saline (PBS) to the 1 mL mark of the tube and heat the tube in a heating block at 95 &#176;C to get 1 mL of a 36% PFA solution. Transfer the 1 mL 36% PFA solution to a tube with a capacity of 15 mL. Add 8 mL PBS to the 1 mL 36% PFA solution, to get 9 mL of a 4% PFA stock solution. Aliquot the 4% PFA stock solution and store at -18 &#176;C until use for fixation of the cells. 
	CAUTION: 1) Potassium hydroxide solution is corrosive. Be aware of eye and skin protection. 2) PFA is carcinogenic. Avoid skin contact and inhalation. Prepare the fixation solution under a hood.</li>
	<li>Permeabilization solution: Add 50 &#181;L Octoxinol 9 to 50 mL PBS to get a solution of approximately 0.1% Octoxinol 9.</li>
	<li>Blocking solution: Prepare 5% and 2% blocking solutions by mixing the protein blocking agent with PBS and store at 6 &#8211;&#160;8 &#176;C.</li>
</ol>
2. Preparation of the Samples
<ol>
	<li>Fibroblasts in cell culture: Cultivate human fibroblasts of the cell line NHDF in a humidified 5% CO2 atmosphere at 37 &#176;C in RPMI 1640 medium supplemented with 10% fetal calf serum, 4 mM glutamine, and 1% penicillin/streptomycin.
	<ol>
		<li>Preparation of a microscope slide with adherent fibroblasts
		<ol>
			<li>Remove the medium from the flask (75 cm2 growth area) with exponentially growing NHDF fibroblasts. Add 10 mL PBS to the flask. Wash the adherent fibroblasts gently by manually shaking the flask for 1 min.</li>
			<li>Remove the PBS and add 1 mL trypsin into the flask. Shake the flask gently to ensure that all adherent cells are covered by trypsin. Remove the trypsin from the flask. Put the flask into the incubator for 5 min at 37 &#176;C.</li>
			<li>Take the flask out of the incubator and add 10 mL RPMI 1640 medium into the flask. Pipette the medium up and down several times to disperse the fibroblasts.</li>
			<li>Pipette 0.3 mL of the dispersed fibroblasts onto each of the two fields of the microscope slide and put the slide into the incubator for 24 h so that the fibroblasts adhere to the slide. For immunostaining of &#947;H2AX and 53BP1 in nuclei of fibroblasts, move on to step 3.1.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Patient samples
	<ol>
		<li>Blood samples: Use the collection tubes that were filled with 2 mL of the anticoagulant stock solution and add 7 mL blood into the tubes. Store the samples overnight at room temperature (i.e., 18 - 20 &#176;C). The next day, isolate the G0/G1 phase resting mononuclear cells by density gradient centrifugation (step 2.2.3).</li>
		<li>Bone marrow samples: Use the collection tubes that were filled with 2 mL of the anticoagulant stock solution (step 1.1) and add 7 mL bone marrow into the tubes. Store the samples overnight at room temperature (i.e.,18 - 20 &#176;C). The next day, isolate the G0/G1 phase resting mononuclear cells by density gradient centrifugation (step 2.2.3). Use CD34 microbeads and cell separation columns for the isolation of the CD34+ cells.</li>
		<li>Isolation of mononuclear cells by density gradient centrifugation 
		NOTE: Mononuclear cells are isolated from blood and bone marrow samples by density gradient centrifugation17,18,19.
		<ol>
			<li>Dilute the heparinized blood sample in a ratio of 1:1 with PBS and the heparinized bone marrow sample in a ratio of 1:3 with PBS. Suspend the cells gently by drawing them in and out of the pipette two times.</li>
			<li>Add a volume of density gradient medium to a fresh tube. Gently layer the diluted blood or bone marrow on top of the density gradient medium. Take care not to mix the two layers.</li>
			<li>Centrifuge the tube for 30 min at room temperature and 400 x g. Draw the upper plasma layer off with the pipette. Then carefully harvest the mononuclear cells in the layer above the density gradient medium. Transfer the mononuclear cells into a fresh tube.</li>
			<li>Add at least three volumes of PBS to the mononuclear cells in the tube. Suspend the cells gently by drawing them in and out of the pipette two times. Centrifuge the tube for 10 min at room temperature and 400 x g.</li>
			<li>After the spin remove the supernatant and add 10 mL of 4 &#176;C, 1x&#160;lysis solution for red cells. Resuspend the cells gently by drawing them in and out of the pipette two times, and place the tube on ice for 5 min. Then add 30 mL PBS at 4 &#176;C and centrifuge the tube for 10 min at room temperature and 400 x g.</li>
		</ol>
		</li>
		<li>Preparation of the cytospins
		<ol>
			<li>Prepare two cytospins with mononuclear cells of the patient samples (i.e., one cytospin for &#947;H2AX immunofluorescence staining and one cytospin for &#947;H2AX and 53BP1 combined immunofluorescence staining) by centrifugation (300 x g, 10 min) of 1.0 x 105 cells for each preparation (Figure 3 and 4).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. &#947;H2AX and 53BP1 Immunofluorescence Staining
<ol>
	<li>Fixation and permeabilization: Fix the cells with 200 &#181;L of 4% PFA for 10 min. After fixation, wash the cells gently three times with 30 mL of PBS for 5 min each on a lab shaker. Then permeabilize the cells with 200 &#181;L of 0.1% Octoxinol 9 for 10 min. Wash the cells gently three times with 30 mL of 5% blocking solution for 5 min each on a lab shaker. Block the cells in 30 mL of fresh 5% blocking solution for 1 h.</li>
	<li>Incubation with antibodies
	<ol>
		<li>Incubate one preparation of the cells with a mouse monoclonal anti-&#947;H2AX antibody (1:500) and the other preparation of the cells with a mouse monoclonal anti-&#947;H2AX antibody (1:500) and a polyclonal rabbit anti-53BP1 antibody (1:500) overnight at 4 &#176;C.</li>
		<li>After incubation wash the cells gently 3 times with 30 mL of 2% blocking solution for each 5 min on a lab shaker.</li>
		<li>Remove the blocking solution and incubate the first preparation of the cells with an Alexa488-conjugated goat anti-mouse secondary antibody (1:500) and the second preparation of the cells with an Alexa488-conjugated goat anti-mouse secondary antibody (1:500) and an Alexa555-conjugated donkey anti-rabbit secondary antibody (1:500) for 1 h at room temperature.</li>
	</ol>
	</li>
	<li>Mounting medium: Put the slide with the cells in a bowl and wash the cells gently three times with 30 mL of PBS for each 5 min on a lab shaker. Remove the PBS and mount the cells with mounting medium containing 4,6-diamidino-2-phenylindole. Cautiously put a coverslip on top of the mounting medium so that no air bubbles are embedded. Wait at least 3 h for hardening of the mounting medium before analyzing the cells by fluorescence microscopy.</li>
</ol>
4. Analysis of &#947;H2AX and 53BP1 Foci
<ol>
	<li>Fluorescence microscopy: Analyse the &#947;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&#160;objective magnification. Record images with a camera and process the images with appropriate imaging software.</li>
</ol>

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<li>Sedelnikova, O. A., Bonner, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((GammaH2AX+in+cancer+cells%3A+a+potential+biomarker+for+cancer+diagnostics%2C+prediction+and+recurrence%5BTitle%5D)+AND+%22Cell+Cycle%22%5BJournal%5D)">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/pubmed?term=((JAK2V617F+promotes+replication+fork+stalling+with+disease-restricted+impairment+of+the+intra-S+checkpoint+response%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">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/pubmed?term=((Increase+of+DNA+damage+and+alteration+of+the+DNA+damage+response+in+myelodysplastic+syndromes+and+acute+myeloid+leukemias%5BTitle%5D)+AND+%22Leuk+Res%22%5BJournal%5D)">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/pubmed?term=((Double-strand+break+repair%3A+53BP1+comes+into+focus%5BTitle%5D)+AND+%22Nat+Rev+Mol+Cell+Biol%22%5BJournal%5D)">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/pubmed?term=((A+cell+cycle-dependent+regulatory+circuit+composed+of+53BP1-RIF1+and+BRCA1-CtIP+controls+DNA+repair+pathway+choice%5BTitle%5D)+AND+%22Mol+Cell%22%5BJournal%5D)">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/pubmed?term=((Separation+of+White+Blood+Cells%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Isolation+of+mononuclear+cells+and+granulocytes+from+human+blood.+Isolation+of+monuclear+cells+by+one+centrifugation%2C+and+of+granulocytes+by+combining+centrifugation+and+sedimentation+at+1+g%5BTitle%5D)+AND+%22Scand+J+Clin+Lab+Invest+Suppl%22%5BJournal%5D)">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>
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<li>Jamur, M. C., Oliver, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Permeabilization+of+cell+membranes%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Permeabilization of cell membranes.</a> Methods Mol Biol. 588, 63-66 (2010).</li>
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The authors have nothing to disclose.

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><tbody><tr><td>RPMI medium</td><td>Sigma-Aldrich</td><td>R0883</td><td>Medium for cell culture</td></tr><tr><td>Heparin sodium</td><td>ratiopharm</td><td>PZN 3029843</td><td>&amp;nbsp;Heparin 5,000 I.U. / mL</td></tr><tr><td>Sodium chloride solution 0.9%</td><td>B. Braun</td><td>PZN 1957154</td><td>Component of the anticoagulant stock solution</td></tr><tr><td>Ficoll-Paque Premium</td><td>GE Healthcare</td><td>17-5442-02</td><td>Medium for isolation of mononuclear cells</td></tr><tr><td>Trypsin solution 10X</td><td>Sigma-Aldrich</td><td>59427C</td><td>Enzyme for dissociation of fibroblasts in cell culture</td></tr><tr><td>CD34 MicroBead Kit</td><td>Miltenyi Biotec</td><td>130-046-702</td><td>Isolation of CD34+ myeloid progenitor cells</td></tr><tr><td>Diagnostic microscope slides</td><td>Thermo Scientific</td><td>ER-203B-CE24</td><td>Microscope slides</td></tr><tr><td>Megafuge 1.0 R</td><td>Heraeus</td><td>75003060</td><td>&amp;nbsp;Tabletop centrifuge&amp;nbsp;</td></tr><tr><td>Cytospin device</td><td></td><td></td><td></td></tr><tr><td>Lid</td><td>Heraeus</td><td>76003422</td><td>Lid for working without micro-tubes</td></tr><tr><td>Cyto container</td><td>Heraeus</td><td>75003416</td><td>Cyto container with 2 conical bores</td></tr><tr><td>Clip carrier</td><td>Heraeus</td><td>75003414</td><td>Carrier for holding a cyto container and a slide</td></tr><tr><td>Support insert</td><td>Heraeus</td><td>75003417</td><td>Support insert for holding a clip carrier</td></tr><tr><td>Triton X-100 (Octoxinol 9)</td><td>Thermo Scientific</td><td>85112</td><td>Detergent for permeabilization of cell membranes</td></tr><tr><td>Potassium hydroxide solution 1M</td><td>Merck Millipore</td><td>109107</td><td>Necessary for preparing the paraformaldehyde solution</td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td>Fixation agent</td></tr><tr><td>Phosphate buffered saline</td><td>Sigma-Aldrich</td><td>D8537</td><td>Balanced salt solution</td></tr><tr><td>Chemiblocker</td><td>Merck Millipore</td><td>2170</td><td>Blocking agent</td></tr><tr><td>Mouse monoclonal anti-&amp;gamma;H2AX antibody (JBW301)</td><td>Merck Millipore</td><td>05-636</td><td>Primary antibody for detection of &amp;gamma;H2AX</td></tr><tr><td>Polyclonal rabbit anti-53BP1 antibody (NB100-304)</td><td>Novus Biologicals</td><td>NB100-304</td><td>Primary antibody for detection of 53BP1</td></tr><tr><td>Alexa Fluor 488-conjugated goat anti-mouse antibody</td><td>Invitrogen</td><td>A-11001</td><td>Secondary antibody</td></tr><tr><td>Alexa Fluor 555-conjugated donkey anti-rabbit antibody</td><td>Invitrogen</td><td>A-31572</td><td>Secondary antibody</td></tr><tr><td>Vectashield mounting medium</td><td>Vector Laboratories</td><td>H-1200</td><td>Contains DAPI for staining of DNA</td></tr><tr><td>Axio Scope.A1</td><td>Zeiss</td><td>490035</td><td>Fluorescence microscope</td></tr><tr><td>Cool Cube 1 CCD camera</td><td>Metasystems</td><td>H-0310-010-MS</td><td>Camera system for digital recording</td></tr><tr><td>Isis software</td><td>Metasystems</td><td>Not applicable</td><td>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 ...

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Immunofluorescence Microscopy of &#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.

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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Research

JoVE Journal

Cancer Research

16.2K Views.  Medical Faculty Mannheim of the Heidelberg University. Dit manuscript biedt een protocol voor de analyse van DNA dubbele-strand einden door immunofluorescentie microscopie van γH2AX en 53BP1.

Video: Immunofluorescence Microscopy of γH2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks

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 ...

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.

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.

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

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 ...

Dual Immunofluorescence of &#947;H2AX and 53BP1 in Human Peripheral Lymphocytes

Double strand breaks (DSBs) are one of the most severe lesions that can occur in cell nuclei, and, if not repaired, they can lead to severe outcomes, including cancer. The cell is, therefore, provided with complex mechanisms to repair DSBs, and these pathways involve histone H2AX in its phosphorylated form at Ser-139 (namely &#947;H2AX) and p53 binding protein 1 (53BP1). As both proteins can form foci at the sites of DSBs, identification of these markers is considered a suitable method to study both DSBs and their kinetics of repair. According to the molecular processes that lead to the formation of &#947;H2AX and 53BP1 foci, it could be more useful to investigate their co-localization near the DSBs in order to set up an alternative approach that allows quantifying DSBs by the simultaneous detection of two DNA damage markers. Thus, this protocol aims to assess the genomic damage induced in human lymphocytes by the radiomimetic agent bleomycin through the presence of &#947;H2AX and 53BP1 foci in a dual immunofluorescence. Using this methodology, we also delineated the variation in the number of &#947;H2AX and 53BP1 foci over time, as a preliminary attempt to study the repair kinetics of bleomycin-induced DSBs.

This protocol presents a method to assess the formation and repair of DNA double-strand breaks through the simultaneous detection of &#947;H2AX and 53BP1 foci in interphase nuclei of bleomycin-treated human peripheral lymphocytes.

Double strand breaks (DSBs) are one of the most severe lesions that can occur in cell nuclei, and, if not repaired, they can lead to severe outcomes, ...

Genetics

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

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

Biology

Quantitation of &gamma;H2AX Foci in Tissue Samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX, is an early response to DNA double-strand breaks1. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Overall, DSB induction results in the formation of discrete nuclear &gamma;H2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2. Given the unique specificity and sensitivity of this marker, analysis of &gamma;H2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of &gamma;H2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, &gamma;H2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of &gamma;H2AX foci ex vivo and in vivo4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of &gamma;H2AX foci in mouse tissues.

Quantitation of &#947;H2AX Foci in Tissue Samples

Quantitation of DNA double-strand breaks on the basis of &gamma;H2AX foci has become an invaluable tool, particularly in radiation biology, for the evaluation of tissue radiosensitivity and effects of radiation modifying compounds. Here we demonstrate the use of an immunofluorescence assay for quantitation of &gamma;H2AX foci in tissue samples.

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) ...

Evaluation of the Spatial Distribution of &gamma;H2AX following Ionizing Radiation

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3. The phosphorylated form of H2AX, referred to as &gamma;H2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3. Analysis and quantitation of &gamma;H2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds4.
Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of &gamma;H2AX as molecular marker of DSBs, a disparity in &gamma;-irradiation-induced &gamma;H2AX foci formation in euchromatin and heterochromatin has been observed5-7. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of &gamma;H2AX following ionizing radiation8. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between &gamma;H2AX formation and active transcription9. Here we demonstrate our immunofluorescence method for detection and quantitation of &gamma;H2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3D-modeling.

Evaluation of the Spatial Distribution of &#947;H2AX following Ionizing Radiation

Microscopic analysis of &gamma;H2AX foci, which form following the phosphorylation of H2AX at Ser-139 in response to DNA double-strand breaks, has become an invaluable tool in radiation biology. Here we used an antibody to mono-methylated histone H3 at lysine 4 as an epigenetic marker of actively transcribing euchromatin, to evaluate the spatial distribution of radiation-induced &gamma;H2AX formation within the nucleus.

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone ...

Visualization of DNA Repair Proteins Interaction by Immunofluorescence

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA insults. Genotoxic agents can damage the DNA backbone, break it, or modify the chemical nature of individual bases. Following DNA insult, DNA damage response (DDR) pathways are activated and proteins involved in the repair are recruited. A plethora of factors are involved in sensing the type of damage and activating the appropriate repair response. Failure to correctly activate and recruit DDR factors can lead to genomic instability, which underlies many human pathologies including cancer. Studies of DDR proteins can provide insights into genotoxic drug response and cellular mechanisms of drug resistance.
There are two major ways of visualizing&#160;proteins in vivo: direct observation, by tagging the protein of interest with a fluorescent protein and following it by live imaging, or indirect immunofluorescence on fixed samples. While visualization of fluorescently tagged proteins allows precise monitoring over time, direct tagging in N- or C-terminus can interfere with the protein localization or function. Observation of proteins in their unmodified, endogenous version is preferred. When DNA repair proteins are recruited to the DNA insult, their concentration increases locally and they form groups, or &#8220;foci&#8221;, that can be visualized by indirect immunofluorescence using specific antibodies.
Although detection of protein foci does not provide a definitive proof of direct interaction, co-localization of proteins in cells indicates that they regroup to the site of damage and can inform of the sequence of events required for complex formation. Careful analysis of foci spatial overlap in cells expressing wild type or mutant versions of a protein can provide precious clues on functional domains important for DNA repair function. Last, co-localization of proteins indicates possible direct interactions that can be verified by co-immunoprecipitation in cells, or direct pulldown using purified proteins.

Following DNA damage, human cells activate essential repair pathways to restore the integrity of their genome. Here, we describe the method of indirect immunofluorescence as a means to detect DNA repair proteins, analyze their spatial and temporal recruitment, and help interrogate protein-protein interaction at the sites of DNA damage.

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA ...

Assessing Protein Interaction and Localization in DNA Damage

Proximity Ligand Assay to Localize Proteins in DNA Damage Sites

The DNA damage response is a genetic information safeguard that protects cells from perpetuating damaged DNA. The characterization of the proteins that cooperate in this process allows the identification of alternative targets for therapeutic intervention in several diseases, such as cancer, aging-related diseases, and chronic inflammation. The Proximity Ligand Assay (PLA) emerged as a tool for estimating interaction between proteins as well as spatial proximity among organelles or cellular structures and allows the temporal localization and co-localization analysis under stress conditions, for instance. The method is simple because it is similar to conventional immunofluorescence and allows the staining of an organelle, cellular structure, or a specific marker such as mitochondria, endoplasmic reticulum, PML bodies, or DNA double-strand marker, yH2AX simultaneously. The phosphorylation of the S139 at Histone 2A variant, H2AX, then referred to as yH2AX, is widely used as a very sensitive and specific marker of DNA double-strand breaks. Each focus of yH2AX staining corresponds to one break in DNA that occurs a few minutes after the damage. The analysis of changes in yH2AX foci is the most common assay for studying if the protein of interest is implicated in DNA damage response (DDR). Whether a direct role in the DNA damage site is expected, fluorescence microscopy is used to verify the colocalization of the protein of interest with yH2AX foci. However, except for the new super-resolution fluorescence methods, to conclude, the local interaction with DNA damage sites can be a little subjective. Here, we show an assay to evaluate the localization of proteins in the DDR pathway using yH2AX as a marker of the damage site. This assay can be used to characterize the temporal localization under different insults that cause DNA damage.

Author Spotlight: Combining Proximity Ligand Assay with Gamma-H2AX Staining to Characterize Protein Interactions in DNA Damage Response

Here, we present a simple and rapid protocol for the detection of protein interaction at DNA damage sites.

The DNA damage response is a genetic information safeguard that protects cells from perpetuating damaged DNA. The characterization of the proteins that ...

Immunofluorescence-Based Visualization of DNA Repair Protein Interactions

Source: de la Pe&#241;a Avalos, B., et al., <a href="https://app.jove.com/v/61447">Visualization of DNA Repair Proteins Interaction by Immunofluorescence.</a> J. Vis. Exp. (2020).
In this video, we describe the immunofluorescence method to visualize the localization of the DNA repair proteins &#947;H2AX and 53BP1 at the sites of double-strand DNA breaks in irradiated cell nuclei.

Source: de la Pe&#241;a Avalos, B., et al., Visualization of DNA Repair Proteins Interaction by Immunofluorescence. J. Vis. Exp. (2020).
In this video, we ...

Immunofluorescence Microscopy of γH2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks

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