Name: Immunofluorescence Microscopy of γH2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks (Video) | JoVE Journal
Uploaded: 2017-11-03T14:00:00.000+00:00
Duration: 10 min 47 s
Description: Learn immunofluorescence microscopy of γH2AX and 53BP1 for analyzing DNA double-strand break formation and repair in cancer research. This method visualizes single breaks and genetic instability mechanisms.

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5 minutes

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>

<ol><li>Hoeijmakers, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome+maintenance+mechanisms+for+preventing+cancer%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Genome maintenance mechanisms for preventing cancer.</a> Nature. 411 (6835), 366-374 (2001).</li>
<li>Lindahl, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Instability+and+decay+of+the+primary+structure+of+DNA%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Instability and decay of the primary structure of DNA.</a> Nature. 362 (6422), 709-715 (1993).</li>
<li>Zeman, M. K., Cimprich, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Causes+and+consequences+of+replication+stress%5BTitle%5D)+AND+%22Nat+Cell+Biol%22%5BJournal%5D)">Causes and consequences of replication stress.</a> Nat Cell Biol. 16 (1), 2-9 (2014).</li>
<li>IAEA. Biological effects of low doses of ionizing radiation: A fuller picture. , 
 <a target="_blank" href="https://www.iaea.org/sites/default/files/36405843745.pdf">https://www.iaea.org/sites/default/files/36405843745.pdf</a> (1994).</li>
<li>Vilenchik, M. M., Knudson, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endogenous+DNA+double-strand+breaks%3A+production%2C+fidelity+of+repair%2C+and+induction+of+cancer%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Endogenous DNA double-strand breaks: production, fidelity of repair, and induction of cancer.</a> Proc Natl Acad Sci U S A. 100 (22), 12871-12876 (2003).</li>
<li>Simsek, D., Jasin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alternative+end-joining+is+suppressed+by+the+canonical+NHEJ+component+Xrcc4-ligase+IV+during+chromosomal+translocation+formation%5BTitle%5D)+AND+%22Nat+Struct+Mol+Biol%22%5BJournal%5D)">Alternative end-joining is suppressed by the canonical NHEJ component Xrcc4-ligase IV during chromosomal translocation formation.</a> Nat Struct Mol Biol. 17 (4), 410-416 (2010).</li>
<li>Weinstock, D. M., Brunet, E., Jasin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Formation+of+NHEJ-derived+reciprocal+chromosomal+translocations+does+not+require+Ku70%5BTitle%5D)+AND+%22Nat+Cell+Biol%22%5BJournal%5D)">Formation of NHEJ-derived reciprocal chromosomal translocations does not require Ku70.</a> Nat Cell Biol. 9 (8), 978-981 (2007).</li>
<li>Rogakou, E. P., Pilch, D. R., Orr, A. H., Ivanova, V. S., Bonner, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DNA+double-stranded+breaks+induce+histone+H2AX+phosphorylation+on+serine+139%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139.</a> J Biol Chem. 273 (10), 5858-5868 (1998).</li>
<li>Scully, R., Xie, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Double+strand+break+repair+functions+of+histone+H2AX%5BTitle%5D)+AND+%22Mutat+Res%22%5BJournal%5D)">Double strand break repair functions of histone H2AX.</a> Mutat Res. 750 (1-2), 5-14 (2013).</li>
<li>Walters, D. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evidence+for+ongoing+DNA+damage+in+multiple+myeloma+cells+as+revealed+by+constitutive+phosphorylation+of+H2AX%5BTitle%5D)+AND+%22Leukemia%22%5BJournal%5D)">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/pubmed?term=((Endogenous+expression+of+phosphorylated+histone+H2AX+in+tumors+in+relation+to+DNA+double-strand+breaks+and+genomic+instability%5BTitle%5D)+AND+%22DNA+Repair+%28Amst%29%22%5BJournal%5D)">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/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>
<li>Bain, B., Pshyk, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enhanced+reactivity+in+mixed+leukocyte+cultures+after+separation+of+mononuclear+cells+on+Ficoll-Hypaque%5BTitle%5D)+AND+%22Transplant+Proc%22%5BJournal%5D)">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/pubmed?term=((Leukocyte+DNA+damage+after+reduced+and+conventional+absorbed+radiation+doses+using+3rd+generation+dual-source+CT+technology%5BTitle%5D)+AND+%22Eur+J+Radiol+Open%22%5BJournal%5D)">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/pubmed?term=((Leukocyte+DNA+damage+after+multi-detector+row+CT%3A+a+quantitative+biomarker+of+low-level+radiation+exposure%5BTitle%5D)+AND+%22Radiology%22%5BJournal%5D)">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/pubmed?term=((In+vivo+formation+and+repair+of+DNA+double-strand+breaks+after+computed+tomography+examinations%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">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/pubmed?term=((Cell+fixatives+for+immunostaining%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">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/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>
<li>Rothkamm, K., Lobrich, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evidence+for+a+lack+of+DNA+double-strand+break+repair+in+human+cells+exposed+to+very+low+x-ray+doses%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">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>

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

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 γH2AX foci in cells is most accurate in the G0/G1 phase and the G2 phase when γH2AX foci appear as distinct fluorescent dots (Figure 5A). In contrast, analysis of γH2AX foci in cells during the S phase is complicated by dispersed pan-nuclear γH2AX speckles caused by the replication process (Figure 5B).
Fixation of the cells was performed with 4% PFA (10 min)...

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

Quantification of DNA double-strand streaks using γH2AX 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 γH2AX foci after exposure of cells to radiation.

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

Quantitation of DNA double-strand breaks on the basis of γH2AX 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 γH2AX foci in tissue samples.

Quantitation of &gamma;H2AX Foci in Tissue Samples

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

In this video, we describe the immunofluorescence method to visualize the localization of the DNA repair proteins γH2AX and 53BP1 at the sites of double-strand DNA breaks in irradiated cell nuclei.

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Immunofluorescence Microscopy for the Analysis of DNA Double-Strand Breaks

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

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.

Visualization of DNA Repair Proteins Interaction by Immunofluorescence

This protocol presents a method to assess the formation and repair of DNA double-strand breaks through the simultaneous detection of γH2AX and 53BP1 foci in interphase nuclei of bleomycin-treated human peripheral lymphocytes.

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

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

Proximity Ligand Assay to Localize Proteins in DNA Damage Sites

Learn immunofluorescence microscopy of γH2AX and 53BP1 for analyzing DNA double-strand break formation and repair in cancer research. This method visualizes single breaks and genetic instability mechanisms.

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

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