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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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. 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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 exponentially growing normal fibroblasts of the cell line NHDF. The &#947;H2AX foci appeared as distinct fluorescent dots in the G0/G1 and G2 phases as compared to the dispersed pan-nuclear &#947;H2AX speckles in S phase (Figure 5). The distinct &#947;H2AX foci in the G0/G1 phase and in G2 phase indicated transient and/or constitutive DNA damage that might have been acquired during repetitive cell cycle passages; conversely, the dispersed speckles in S phase occurred only transiently and were presumably associated with the replication process. As the pan-nuclear &#947;H2AX speckles may disturb the analysis of genuine &#947;H2AX foci, the exclusion of S phase cells from analysis is recommended, which is readily done by the S phase specific &#947;H2AX pattern. Alternatively, cells in the S phase can be excluded by measurement of the intensity of the DAPI signal or by application of S phase specific markers (e.g., Cyclin A). Moreover, the induction of growth arrest with synchronization of cells in G0/G1 phase may be useful before preparing the cytospins by storing the samples at room temperature (i.e., 18 - 20 &#176;C) overnight.
The procedures of fixation and permeabilization are critical steps in immunostaining of &#947;H2AX and 53BP1. Fixation is necessary to preserve the morphology and antigenicity of the cells23. Permeabilization enables access to the intracellular and nuclear antigens24. PFA is a relatively gentle agent for fixation and stabilizes cells while preserving protein structures by reacting with basic amino acids to form methylene crosslinks. After fixation with PFA, cytospins can be stored for days and weeks until further processing. For detection of &#947;H2AX and 53BP1, the cells have to be permeabilized. Octoxinol 9 was used as a non-ionic detergent containing uncharged hydrophilic head groups consisting of polyoxyethylene moieties. Octoxinol 9 breaks up the cell nuclei gently and preserves &#947;H2AX foci quite distinctly. Alternatively, methanol can be used for the fixation and permeabilization of the cells, too. Methanol precipitates proteins and dissolves lipids from the cell membrane, making them permeable to antibodies. However, treatment with methanol is presumably more aggressive and &#947;H2AX foci may not appear as distinctly when compared to the &#947;H2AX foci obtained by treatment with 4% PFA and 0.1% Octoxinol 9. Notwithstanding, efficient antibodies play a key role for the immunostaining of &#947;H2AX and 53BP1. Therefore, the use of proven antibodies is recommended (see Table of Materials/Equipment). A serial dilution of the antibodies may be useful for determination of the optimal antibody concentrations.
&#947;H2AX foci are used as a marker in radiation biodosimetry at low doses &#62; 1 mGy25. &#947;H2AX foci were analyzed in lymphocytes at 5 min after x-ray irradiation with a dose of 100 mGy in vitro (Figure 6). As H2AX is phosphorylated rapidly at sites of DSB after irradiation, the number of &#947;H2AX foci may already reach a maximum at 5 min after irradiation20,22. However, the repair process starts instantly after irradiation and &#947;H2AX foci are removed at a distinct decay rate. Hence, for monitoring the number of &#947;H2AX foci in a chronological sequence, it is advisable to interrupt the repair process by putting the sample on ice&#160;at the specified point in time&#160;after irradiation.
Immunofluorescence microscopy of &#947;H2AX and 53BP1 is a useful method for analyzing genetic instability in cancer cells. CD34+ cells were enriched from the bone marrow of a patient with acute myeloid leukemia using CD34 microbeads. Images of &#947;H2AX foci and 53BP1 foci were obtained using specific fluorescence filters. A bright illumination of the cytospin is important to induce adequate fluorescence of the &#947;H2AX and 53BP1 foci. After recording the image, adaption of the intensity of the foci and the background signal is necessary using the imaging software. For analyzing the co-localization of &#947;H2AX and 53BP1 foci, the images are merged (Figure 7). A basic epifluorescence microscope equipped with filters for DAPI, Alexa 488, and Cy3 in combination with a camera system and imaging software is sufficient for the analysis of &#947;H2AX and 53BP1 foci in DAPI stained cell nuclei. However, certain applications (e.g., analysis of DNA damage along single particle tracks) may require higher resolution and reduction of the background fluorescence (as provided e.g., by confocal laser scanning microscopy). Moreover, microscope technology is a rapidly developing field and a resolution below 200 nm is feasible with state-of-the-art microscopes.
In summary, this manuscript highlights critical issues of DSB formation and repair and provides a step-by-step protocol for a detailed analysis of DSB by immunofluorescence microscopy of &#947;H2AX and 53BP1. The research on DSB will clearly benefit from recent and future developments in microscope technology.

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 with 4% PFA (10 min) and permeabilization with 0.1% Octoxinol 9 (10 min). Methanol may be used for fixation and permeabilization, too; however, methanol may break up the cell nuclei quite severely and smear out the &#947;H2AX foci so the &#947;H2AX foci may not appear distinct when compared to the &#947;H2AX foci obtained by treatment with 4% PFA and 0.1% Octoxinol 9.
The choice of efficient antibodies is critical for effective immunofluorescence microscopy. Experiments were performed with a primary mouse monoclonal anti-&#947;H2AX antibody and a primary rabbit polyclonal anti-53BP1 antibody for immunofluorescence staining of &#947;H2AX and 53BP1, respectively.
As each &#947;H2AX focus is considered to represent a single DSB, immunofluorescence staining of &#947;H2AX can be used for the quantification of DSB in irradiated tissues. Levels of &#947;H2AX foci in irradiated lymphocytes (Figure 6) are proportional to the irradiation dose20,21. &#947;H2AX foci may achieve a maximum already 5 min of irradiation20,22. The decay of &#947;H2AX foci at later points in time can be monitored and reflects the repair of DSB20,21,22.
Genetic instability can be analyzed by combined immunofluorescence staining of &#947;H2AX and 53BP1 in cell lines and tumor specimens10,11,12,13,14. An exemplary analysis was performed in the CD34+ cells of a patient with acute myeloid leukemia (Figure 7). Co-localization of &#947;H2AX and 53BP1 indicated promotion of 53BP1-mediated NHEJ repair mechanisms at sites of DSB while HR was prevented. However, the percentage of co-localizing &#947;H2AX and 53BP1 foci in blasts of acute myeloid leukemia was variable. The &#947;H2AX foci without an additional 53BP1 signal might have engaged HR for the repair of DSB in late S/G2 phase. The role of singular 53BP1 foci remained elusive.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56617/56617fig1v3.jpg" /> 
Figure 1: Sources and consequences of endogenous and exogenous DNA damage1,2,3,4. DSB, DNA double-strand breaks; NHEJ, nonhomologous end-joining.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56617/56617fig2v2.jpg" /> 
Figure 2: Schematic view of the major DNA double-strand break (DSB) repair mechanisms in mammalian cells. Homologous recombination uses an intact sister chromatid in late S/G2 phase for potentially error-free repair of DSB. Nonhomologous end-joining (NHEJ) is active throughout the cell cycle and potentially error-prone as few base pairs may be added or resected before ligation of the DSB ends. Alternative end-joining is a slow mutagenic back-up repair mechanism which might be engaged in the case of NHEJ deficiency. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56617/56617fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56617/56617fig3v2.jpg" /> 
Figure 3: Device for preparing the cytospins. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56617/56617fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56617/56617fig4v2.jpg" /> 
Figure 4: Preparation of the cytospins.Two cytospins of each sample are prepared by centrifugation of 1.0 x 105 cells at 300 x g for 10 min. One cytospin is used for &#947;H2AX immunofluorescence staining and another cytospin is utilized for combined &#947;H2AX and 53BP1 immunofluorescence staining. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56617/56617fig4v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56617/56617fig5v2.jpg" /> 
Figure 5: Cell cycle dependent patterns of &#947;H2AX foci in normal fibroblasts of the cell line NHDF. (A) &#947;H2AX foci (green, Alexa 488) in nuclei (blue, DAPI) of NHDF fibroblasts appear as distinct dots in G0/G1 or G2 phase. (B) &#947;H2AX foci in nuclei of fibroblasts in S phase appear as dispersed pan-nuclear speckles (scale bar = 5 &#181;m). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56617/56617fig5v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56617/56617fig6v3.jpg" /> 
Figure 6: &#947;H2AX foci in x-ray irradiated lymphocytes of a healthy individual. The images of &#947;H2AX foci (green, Alexa 488) in nuclei (blue, DAPI) of lymphocytes are recorded at 5 min after irradiation with 100 mGy (scale bar = 5 &#181;m). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56617/56617fig6v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56617/56617fig7.jpg" /> 
Figure 7: Co-localization of &#947;H2AX and 53BP1 foci in CD34+ cells of a patient with acute myeloid leukemia. Co-localization of &#947;H2AX foci (green, Alexa 488) and 53BP1 foci (red, Cy3) indicates promotion of 53BP1-mediated nonhomologous end-joining repair mechanisms at sites of DNA double-strand breaks (scale bar = 5 &#181;m). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56617/56617fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Immunofluorescence Microscopy of &#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 ...

immunofluorescence-microscopy-h2ax-53bp1-for-analyzing-formation

Nanyang Technological University

Research

JoVE Journal

Cancer Research

16.1K Views.  Medical Faculty Mannheim of the Heidelberg University. This manuscript provides a protocol for the analysis of DNA double-strand breaks by immunofluorescence microscopy of γH2AX and 53BP1. 

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

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 ...

In Vivo Immunofluorescence Localization for Assessment of Therapeutic and Diagnostic Antibody Biodistribution in Cancer Research

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in tumorigenesis, can be directed to cancer biomarkers enabling tumor detection and characterization, and can be used for cancer therapy as mAbs or antibody-drug conjugates to activate immune effector cells, to inhibit signaling pathways, or directly kill cells carrying the specific antigen. Despite clinical advancements in the development and production of novel and highly specific mAbs, diagnostic and therapeutic applications can be impaired by the complexity and heterogeneity of the tumor microenvironment. Thus, for the development of efficient antibody-based therapies and diagnostics, it is crucial to assess the biodistribution and interaction of the antibody-based conjugate with the living tumor microenvironment. Here, we describe In Vivo Immunofluorescence Localization (IVIL) as a new approach to study interactions of antibody-based therapeutics and diagnostics in the in vivo physiological and pathological conditions. In this technique, a therapeutic or diagnostic antigen-specific antibody is intravenously injected in&#160;vivo and localized ex vivo with a secondary antibody in isolated tumors. IVIL, therefore, reflects the in vivo biodistribution of antibody-based drugs and targeting agents. Two IVIL applications are described assessing the biodistribution and accessibility of antibody-based contrast agents for molecular imaging of breast cancer. This protocol will allow future users to adapt the IVIL method for their own antibody-based research applications.

The in vivo immunofluorescence localization (IVIL) method can be used to examine in vivo biodistribution of antibodies and antibody conjugates for oncological purposes in living organisms using a combination of in vivo tumor targeting and ex vivo immunostaining methods.

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in ...

Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody

Monoclonal antibodies are high affinity multifunctional drugs that work by variable independent mechanisms to eliminate cancer cells. Over the last few decades, the field of antibody-drug conjugates, bispecific antibodies, chimeric antigen receptors (CAR) and cancer immunotherapy has emerged as the most promising area of basic and therapeutic investigations. With numerous successful human trials targeting immune checkpoint receptors and CAR-T cells in leukemia and melanoma at a breakthrough pace, it is highly exciting times for oncologic therapeutics derived from variations of antibody engineering. Regrettably, a significantly large numbers of antibody and CAR based therapeutics have also proven disappointing in human trials of solid cancers because of the limited availability of immune effector cells in the tumor bed. Importantly, nonspecific distribution of therapeutic antibodies in tissues other than tumors also contribute to the lack of clinical efficacy, associated toxicity and clinical failure. As faithful translation of preclinical studies into human clinical trails are highly relied on mice tumor xenograft efficacy and safety studies, here we highlight a method to test the tumor and general tissue distribution of therapeutic antibodies. This is achieved by labeling the protein-A purified antibody with near Infrared fluorescent dye followed by live imaging of tumor bearing mice.

Here we present a protocol to study the in vivo localization of antibodies in mice tumor xenograft models.

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

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

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

Quantifying Replication Stress in Ovarian Cancer Cells Using Single-Stranded DNA Immunofluorescence

Replication stress is a hallmark of several ovarian cancers. Replication stress can emerge from multiple sources, including double-strand breaks, transcription-replication conflicts, or amplified oncogenes, inevitably resulting in the generation of single-stranded DNA (ssDNA). Quantifying ssDNA, therefore, presents an opportunity to assess the level of replication stress in different cell types and under various DNA-damaging conditions or treatments. Emerging evidence also suggests that ssDNA can be a predictor of responses to chemotherapeutic drugs that target DNA repair. Here, we describe a detailed immunofluorescence-based methodology to quantify ssDNA. This methodology involves labeling the genome with a thymidine analog, followed by the antibody-based detection of the analog at the chromatin under non-denaturing conditions. Stretches of ssDNA can be visualized as foci under a fluorescence microscope. The number and intensity of the foci directly co-relate with the level of ssDNA present in the nucleus. We also describe an automated pipeline to quantify the ssDNA signal. The method is rapid and reproducible. Furthermore, the simplicity of this methodology makes it amenable to high-throughput applications such as drug and genetic screens.

Here, we describe an immunofluorescence-based method to quantify the levels of single-stranded DNA in cells. This efficient and reproducible method can be utilized to examine replication stress, a common feature in several ovarian cancers. Additionally, this assay is compatible with an automated analysis pipeline, which further increases its efficiency.