Name: Dual Immunofluorescence of γH2AX and 53BP1 in Human Peripheral Lymphocytes (Video) | JoVE Journal
Uploaded: 2023-07-14T12:00:00.000+00:00
Duration: 5 min 34 s
Description: Learn dual immunofluorescence of γH2AX and 53BP1 to assess DNA double-strand break formation and repair in bleomycin-treated human peripheral lymphocytes. This method enables precise quantification of genomic damage.

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5 minutes

We are grateful to the whole blood donors and all the health personnel who took the blood samples.

The study was approved by the ethical committee of Pisa University, and informed and signed consent was obtained from each donor.
1. Formation of &#947;H2AX and 53BP1 foci
<ol>
	<li>Preparation of samples and mutagenic treatment
	<ol>
		<li>Collect whole blood samples by venipuncture from healthy adult individuals in blood collection (e.g., Vacutainer) tubes containing lithium heparin as an anticoagulant.</li>
		<li>In order to guarantee proper blood sample preservation, start the procedure within 24 h of sampling.</li>
		<li>Add 300 &#181;L of the sample to a tube containing 4.7 mL of complete medium (0.5% penicillin-streptomycin, 0.75% phytohemagglutinin, 10% FBS previously inactivated, 88.75% RPMI 1640).</li>
		<li>Then add bleomycin sulfate to a final concentration of 5 &#181;g/mL. 
		CAUTION: Bleomycin sulfate is a mutagen. Avoid skin contact and inhalation. Prepare the solution and add the sample under a hood.</li>
		<li>For each sample, set up a negative control (absence of mutagen).</li>
		<li>Place the tube in a thermostat at 37 &#176;C for 2 h.</li>
	</ol>
	</li>
	<li>Fixation
	<ol>
		<li>Centrifuge samples at 540 x g for 5 min at room temperature.</li>
		<li>Aspirate the supernatant and resuspend the pellet with vortex.</li>
		<li>Add 5 mL of hypotonic solution (2.87 g of KCl dissolved in 500 mL of deionized water) and 400 &#181;L of pre-fixative solution (5:3 acetic acid: methanol) to cause hemolysis of red blood cells.</li>
		<li>Centrifuge samples at 540 x g for 5 min at room temperature.</li>
		<li>Aspirate the supernatant and resuspend the pellet in 5 mL of methanol at room temperature for at least 30 min to fix the cells.</li>
		<li>Alternatively, store the cells at -20 &#176;C until use.</li>
	</ol>
	</li>
	<li>Slides preparation
	<ol>
		<li>Centrifuge samples at 540 x g for 5 min at room temperature.</li>
		<li>Aspirate the supernatant and resuspend the pellet in 5 mL of a 3:1 methanol: acetic acid solution. Repeat these steps one more time.</li>
		<li>At the end, centrifuge the solution again at 540 x g for 5 min at room temperature.</li>
		<li>Aspirate the supernatant again leaving enough solution (0.5 mL) to resuspend the pellet, pipette vigorously, drop the resuspended cell pellet on the slides, and air dry. Store the slides at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Immunofluorescence 
	NOTE: Immunofluorescence is an immunologic method for identifying specific cell targets using a primary antibody binding the target itself and a fluorescent secondary antibody binding the primary that allows localizing the target11. In this case, a mouse monoclonal anti-53BP1 (1:50) and a rabbit polyclonal anti-&#947;H2AX (1:50) are used as primary antibodies, while AlexaFluor568 anti-mouse (1:400) and DyLight488 anti-rabbit (1:200) are used as secondary antibodies, respectively.
	<ol>
		<li>Wash the slides two times in 50 mL of 1x PBS for 5 min in couplin jar (16 slides back-to-back).</li>
		<li>Then keep them for 30 min in blocking solution (10 mL of FBS, 10 mL of 10x PBS, 80 mL of deionized water, 300 &#181;L of Triton-X).</li>
		<li>Add to each slide 10 &#181;L of each of the two solutions containing the primary antibodies dissolved in the blocking solution. Cover the slides with paraffin tape and incubate at 4 &#176;C overnight.</li>
		<li>After incubation, perform three washing in 1x PBS for 5 min.</li>
		<li>Add to each slide 10 &#181;L of each of the two solutions containing the secondary antibodies dissolved in the blocking solution. Cover the slides with paraffin tape and incubate at room temperature for 2 h.</li>
		<li>Perform three washing in 1x PBS for 5 min.</li>
		<li>Add 2.5 &#181;L of antifade solution with DAPI on the coverslips before the assembly to counterstain the nuclei. 
		&#8203;NOTE: In order to assess foci kinetics, the procedure is the same as described from 1.1 to 1.4, noting that cell harvesting and the dual immunofluorescence are performed at 0 h, after 2 h post-bleomycin treatment and then, after having removed the mutagen, at 4 h, 6 h, and 24 h post-treatment.</li>
	</ol>
	</li>
</ol>
2. Analysis through a fluorescence microscope
NOTE: &#34;Fluorescence microscope&#34; refers to any microscope that uses fluorescence to generate an image. The specimen is illuminated with light of a specific wavelength (or wavelengths) which is absorbed by the fluorophores, causing them to emit light of longer wavelengths12. AlexaFluor568 and DyLight 488 absorb light of approximately 568 and 488 nm and emit light of 603 and 520 nm, respectively. Thus, they are visible as red or green fluorescence using a TRITC or a FITC filter, respectively.
<ol>
	<li>Score the presence of foci in each slide under a 100x immersion objective (Figure 1).</li>
	<li>Score 200 nuclei per slide and count the number of &#947;H2AX/53BP1 foci in each nucleus.</li>
	<li>Express results in terms of the average number of foci per total nuclei scored. These include both &#947;H2AX/53BP1 positive (showing at least one fluorescence signal) and negative (not showing any fluorescence signal) nuclei.</li>
</ol>

<ol><li>Chatterjee, N., Walker, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanisms+of+DNA+damage%2C+repair+and+mutagenesis%5BTitle%5D)+AND+%22Environmental+and+Molecular+Mutagenesis%22%5BJournal%5D)">Mechanisms of DNA damage, repair and mutagenesis.</a> Environmental and Molecular Mutagenesis. 58 (5), 235-263 (2017).</li>
<li>Aleksandrov, R., Hristova, R., Stoynov, S., Gospodinov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+chromatin+response+to+double-strand+DNA+breaks+and+their+repair%5BTitle%5D)+AND+%22Cells%22%5BJournal%5D)">The chromatin response to double-strand DNA breaks and their repair.</a> Cells. 9 (8), 1853(2020).</li>
<li>Jackson, S. P., Bartek, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+DNA-damage+response+in+human+biology+and+disease%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">The DNA-damage response in human biology and disease.</a> Nature. 461 (7267), 1071-1078 (2009).</li>
<li>Dickey, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((H2AX%3A+functional+roles+and+potential+applications%5BTitle%5D)+AND+%22Chromosoma%22%5BJournal%5D)">H2AX: functional roles and potential applications.</a> Chromosoma. 118 (6), 683-692 (2009).</li>
<li>Her, J., Bunting, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((How+cells+ensure+correct+repair+of+DNA+double-strand+break%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%2C+Thematic+Minireview%22%5BJournal%5D)">How cells ensure correct repair of DNA double-strand break.</a> Journal of Biological Chemistry, Thematic Minireview. 293 (27), 10502-10511 (2018).</li>
<li>Kuo, L. K., Yang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((%CE%B3-H2AX+-+A+novel+biomarker+for+DNA+double-strand+breaks%5BTitle%5D)+AND+%22In+Vivo%22%5BJournal%5D)">γ-H2AX - A novel biomarker for DNA double-strand breaks.</a> In Vivo. 22 (3), 305-310 (2008).</li>
<li>Bártová, E., Legartova, S., Dundr, M., Suchánková, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+role+of+the+53BP1+protein+in+genome+protection%3A+structural+and+functional+characteristics+of+53BP1-dependent+DNA+repair%5BTitle%5D)+AND+%22Aging%22%5BJournal%5D)">A role of the 53BP1 protein in genome protection: structural and functional characteristics of 53BP1-dependent DNA repair.</a> Aging. 11 (8), 2488-2511 (2019).</li>
<li>Popp, H. D., Brendel, S., Hofman, W., Fabarius, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunofluorescence+microscopy+of+%CE%B3H2AX+and+53BP1+for+analyzing+the+formation+and+repair+of+DNA+double-strand+breaks%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Immunofluorescence microscopy of γH2AX and 53BP1 for analyzing the formation and repair of DNA double-strand breaks.</a> Journal of Visualized Experiments. (129), 56617(2017).</li>
<li>Jezkova, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Particles+with+similar+LET+values+generate+DNA+breaks+of+different+complexity+and+reparability%3A+a+high-resolution+microscopy+analysis+of+%CE%B3H2AX%2F53BP1+foci%5BTitle%5D)+AND+%22Nanoscale%22%5BJournal%5D)">Particles with similar LET values generate DNA breaks of different complexity and reparability: a high-resolution microscopy analysis of γH2AX/53BP1 foci.</a> Nanoscale. 10, 1162-1179 (2018).</li>
<li>Scarpato, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kinetics+of+nuclear+phosphorylation+%28%CE%B3-H2AX%29+in+human+lymphocytes+treated+in+vitro+with+UVB%2C+bleomycin+and+mitomycin+C%5BTitle%5D)+AND+%22Mutagenesis%22%5BJournal%5D)">Kinetics of nuclear phosphorylation (γ-H2AX) in human lymphocytes treated in vitro with UVB, bleomycin and mitomycin C.</a> Mutagenesis. 28 (4), 465-473 (2013).</li>
<li>Im, K., Mareninov, S., Diaz, M. F. P., Yong, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+introduction+to+performing+immunofluorescence+staining%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">An introduction to performing immunofluorescence staining.</a> Methods in Molecular Biology. 1897, 299-311 (2019).</li>
<li>Sanderson, M. J., Smith, I., Parker, I., Bootman, M. D. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aSanderson%2C+MJ+%22Fluorescence+microscopy%22">Fluorescence microscopy</a>. 10, Cold Spring Harbor Protocol. (2014).</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+in+Molecular+Biology%22%5BJournal%5D)">Cell fixatives for immunostaining.</a> Methods in Molecular Biology. , 55-61 (2010).</li>
<li>Jamur, M. C., Oliver, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Permeabilization+of+the+cell+membrane%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Permeabilization of the cell membrane.</a> Methods in Molecular Biology. 588, 63-66 (2010).</li>
<li>Hecht, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bleomycin%3A+New+perspectives+on+the+mechanism+of+action%5BTitle%5D)+AND+%22Journal+of+Natural+Products%22%5BJournal%5D)">Bleomycin: New perspectives on the mechanism of action.</a> Journal of Natural Products. 63, 158-168 (2000).</li>
<li>Fei, P., El-Deiry, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((P53+and+radiation+responses%5BTitle%5D)+AND+%22Oncogene%22%5BJournal%5D)">P53 and radiation responses.</a> Oncogene. 22, 5774-5783 (2003).</li>
<li>Mahaney, B. L., Meek, K., Lees-Miller, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Repair+of+ionizing+radiation-induced+DNA+double-strand+breaks+by+non-homologous+end-joining%5BTitle%5D)+AND+%22Biochemical+Journal%22%5BJournal%5D)">Repair of ionizing radiation-induced DNA double-strand breaks by non-homologous end-joining.</a> Biochemical Journal. 417 (3), 639-650 (2009).</li>
<li>Palla, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gamma-H2AX%3A+Can+it+be+established+as+a+classical+cancer+prognostic+factor%5BTitle%5D)+AND+%22Tumor+Biology%22%5BJournal%5D)">Gamma-H2AX: Can it be established as a classical cancer prognostic factor.</a> Tumor Biology. 39 (3), 1010428317695931(2017).</li>
<li>Markovà, E., Hillert, L., Malmgren, L., Persson, B. R. R., Belyaev, I. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microwaves+from+GSM+mobile+telephones+affect+53BP1+and+gamma-H2AX+foci+in+human+lymphocytes+from+hypersensitive+and+healthy+persons%5BTitle%5D)+AND+%22Environmental+Health+Perspectives%22%5BJournal%5D)">Microwaves from GSM mobile telephones affect 53BP1 and gamma-H2AX foci in human lymphocytes from hypersensitive and healthy persons.</a> Environmental Health Perspectives. 113 (9), 1172-1177 (2005).</li>
<li>Scarpato, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nuclear+damage+in+peripheral+lymphocytes+of+obese+and+overweight+Italian+children+as+evaluated+by+the+%CE%B3-H2AX+focus+assay+and+micronucleus+test%5BTitle%5D)+AND+%22The+FASEB+Journal%22%5BJournal%5D)">Nuclear damage in peripheral lymphocytes of obese and overweight Italian children as evaluated by the γ-H2AX focus assay and micronucleus test.</a> The FASEB Journal. 25 (2), 685-693 (2018).</li>
<li>Shanbhag, N. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Early+neuronal+accumulation+of+DNA+double-strand+breaks+in+Alzheimer%27s+disease%5BTitle%5D)+AND+%22Acta+Neuropathologica+Communication%22%5BJournal%5D)">Early neuronal accumulation of DNA double-strand breaks in Alzheimer's disease.</a> Acta Neuropathologica Communication. 7 (1), 77(2019).</li>
<li>Lassmann, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+formation+of+gamma-H2AX+and+53BP1+DNA+repair+foci+in+blood+cells+after+radioiodine+therapy+of+differentiated+thyroid+cancer%5BTitle%5D)+AND+%22Journal+of+Nuclear+Medicine%22%5BJournal%5D)">In vivo formation of gamma-H2AX and 53BP1 DNA repair foci in blood cells after radioiodine therapy of differentiated thyroid cancer.</a> Journal of Nuclear Medicine. 51 (8), 1318-1325 (2010).</li>
<li>Derlin, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assessment+of+%CE%B3-H2AX+and+53BP1+foci+in+peripheral+blood+lymphocytes+to+predict+subclinical+hematotoxicity+and+response+in+somatostatin+receptor-targeted+radionuclide+therapy+for+advanced+gastroenteropancreatic+neuroendocrine+tumors%5BTitle%5D)+AND+%22Cancers+%28Basel%29%22%5BJournal%5D)">Assessment of γ-H2AX and 53BP1 foci in peripheral blood lymphocytes to predict subclinical hematotoxicity and response in somatostatin receptor-targeted radionuclide therapy for advanced gastroenteropancreatic neuroendocrine tumors.</a> Cancers (Basel). 13 (7), 1516(2021).</li>
<li>Djuzenova, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Radiosensitivity+in+breast+cancer+assessed+by+the+histone+%CE%B3-H2AX+and+53BP1+foci%5BTitle%5D)+AND+%22Radiation+Oncology%22%5BJournal%5D)">Radiosensitivity in breast cancer assessed by the histone γ-H2AX and 53BP1 foci.</a> Radiation Oncology. 24, 8-98 (2013).</li>
<li>Atkinson, J., Bezak, E., Kempson, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+DNA+double-strand+breaks+-+are+we+there+yet%5BTitle%5D)+AND+%22Nature+Reviews+in+Molecular+Cell+Biology%22%5BJournal%5D)">Imaging DNA double-strand breaks - are we there yet.</a> Nature Reviews in Molecular Cell Biology. 23, 579-580 (2022).</li>
</ol>

The authors have nothing to disclose.

Immunofluorescence analysis of &#947;H2AX and 53BP1 foci is a suitable method for assessing genomic damage in interphase nuclei of a cell system. This procedure has several critical points that can affect the outcome of the experiments, mainly, the agents used in fixation and permeabilization, the type of antibodies and their dilution factors, and the concentration of the mutagen.
The maintenance of protein integrity is fundamental since the immunofluorescence method expects to identify antige...

DNA damage is continuously induced by agents that can be endogenous, such as ROS generated by cellular oxidative metabolism, or exogenous, both chemicals and physical1. Among the most harmful lesions, double-strand breaks (DSBs) play a fundamental role in contributing to genomic instability, since they cause chromosome aberrations that in turn can initiate the carcinogenesis process. Thus, cells are provided with complex and efficient mechanisms of DSBs repairing2.
When a DSB occurs, the cell triggers the DNA damage response (DDR) where, together with the MRE11/RAD50/NBS1 complex, ATM or ATR kinases are recruited to activate other proteins that slow down or stop the cell cycle3. An essential target of these kinases is histone H2AX, which is phosphorylated on Ser-139 within a few megabases from the DSBs (namely &#947;H2AX), thereby allowing the recruitment of several repair factors such as, among others, BRCA1 and p53 binding protein 1 (53BP1)3. Later, one pathway among homologous recombination (HR), non-homologous end joining (NHEJ), or single-strand annealing (SSA) is triggered to repair the DSBs4,5. Therefore, 53BP1 is involved in dictating the choice between HR or NHEJ, mainly promoting the activation of NHEJ rather than HR6. Moreover, both the phosphorylated form of H2AX histone and 53BP1 can form foci at the sites of DSBs. As these foci persist until the integrity of the double strand is restored, assessing the appearance/disappearance of &#947;H2AX or 53BP1 foci within a time interval is considered a useful method to evaluate the occurrence and repair of DSBs in a cell system6,7. However, according to the molecular processes above described, since &#947;H2AX and 53BP1 foci are expected to co-localize near the DSBs during DDR8,9, it can be useful to detect concurrently the presence of these markers in a dual immunofluorescence.
Thus, the aim of this manuscript was to evaluate the suitability of the simultaneous quantification of &#947;H2AX and 53BP1 foci to assess the genomic damage induced in human peripheral lymphocytes by the radiomimetic agent bleomycin. Using the same methodology, we also attempted to delineate repair kinetics of bleomycin-induced DSBs according to a previously set up experimental procedure10.

<table><tbody><tr><td>AlexaFluor 568 goat anti-mouse IgG (&amp;gamma;1)</td><td>Invitrogen</td><td>A21124</td><td>53BP1 secondary antibody</td></tr><tr><td>Bleoprim</td><td>Sanofi</td><td></td><td>bleomycin sulfate (mutagen)</td></tr><tr><td>Penicillin-streptomycin solution 100X</td><td>Euroclone</td><td>ECB3001D</td><td>antibiotics for culture medium</td></tr><tr><td>PBS 10X</td><td>Termofisher</td><td>14200075</td><td>Phosphate-buffered saline</td></tr><tr><td>FBS</td><td>Euroclone</td><td>EC20180L</td><td>Fetal Bovine Serum for immunofluorescence</td></tr><tr><td>Goat anti-rabbit IgG (H+L) DyLight 488 Coniugated</td><td>Termofisher</td><td>#35552</td><td>&amp;gamma;H2AX secondary antibody</td></tr><tr><td>Mouse anti-53BP1 monoclonal antibody</td><td>Merck</td><td>MAB 3802</td><td>53BP1 primary antibody</td></tr><tr><td>Labophot 2</td><td>Nikon</td><td></td><td>Fluorescence microscope</td></tr><tr><td>P-histone H2AX (Ser139) rabbit antibody</td><td>Cell Signaling</td><td>#2577</td><td>&amp;gamma;H2AX primary antibody</td></tr><tr><td>Phytohemoagglutinin</td><td>Termofisher</td><td>R30852801</td><td>component of culture medium</td></tr><tr><td>Prolong gold antifade reagent with DAPI</td><td>Cell Signaling</td><td>#8961</td><td>Antifade solution with DAPI for counterstaining</td></tr><tr><td>RPMI 1640</td><td>Euroclone</td><td>ECB9006L</td><td>Culture medium</td></tr><tr><td>Triton-X100</td><td>Sigma</td><td>T9284</td><td>Nonionic detergent for permeabilization</td></tr></tbody></table>

dual immunofluorescence of &#947;h2ax and 53bp1 in human peripheral lymphocytes

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.

Data obtained by the fluorescence microscope analysis of peripheral lymphocytes allow us to evaluate three main aspects: the effectiveness of bleomycin treatment in increasing the number of γH2AX and 53BP1 foci (and thus of DSBs) due to its mutagenic effect, at what extent both foci co-localized at the site of DSBs, and the time-course of γH2AX and 53BP1 foci to delineate the repair kinetics of bleomycin-induced DSBs. As expected, a very higher frequency of both γH2AX and 53BP1 foci was observed between untreated and tre...

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, ...

dual-immunofluorescence-h2ax-53bp1-human-peripheral

Research

JoVE Journal

Genetics

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

Microscopic analysis of γH2AX 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 γH2AX formation within the nucleus.

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

In this video, we describe the immunofluorescence microscopy technique to detect specific DNA damage response proteins localized at the sites of DNA double-strand breaks in human mononuclear cells. The proteins are recognized by specific primary antibodies, which in turn bind to fluorophore-tagged secondary antibodies that fluoresce during microscopic visualization, enabling visualization of the proteins as distinct fluorescent foci within the cell nuclei.

Immunofluorescence Microscopy for the Analysis of DNA Double-Strand Breaks

This manuscript provides a protocol for the analysis of DNA double-strand breaks by immunofluorescence microscopy of γH2AX and 53BP1.

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

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.

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

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.

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 the slide preparation and automated scoring of the γ-H2AX foci assay on peripheral blood lymphocytes. To illustrate the method and sensitivity of the assay, isolated lymphocytes were irradiated in vitro. This automated method of DNA DSB detection is useful for fast and high-throughput biological dosimetry applications.

An Automated Microscopic Scoring Method for the &#947;-H2AX Foci Assay in Human Peripheral Blood 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 dual immunofluorescence of γH2AX and 53BP1 to assess DNA double-strand break formation and repair in bleomycin-treated human peripheral lymphocytes. This method enables precise quantification of genomic damage.

Dual Immunofluorescence of γH2AX and 53BP1 in Human Peripheral Lymphocytes

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