We thank the Flow Cytometry Facility team at the German Cancer Research Center (DKFZ) for their support.

1. Preparation
<ol>
	<li>Prepare &#8805;1 x 105 cells/sample in any type of culture vessel as starting material.
	<ol>
		<li>For example, conduct a time-course experiment after exposure of U87 glioblastoma cells to ionizing radiation: Irradiate sub-confluent U87 cells in T25 flasks in triplicates for each time point. Choose early time-points (15 min up to 8 h after irradiation) to follow the kinetics of DSB repair (&#947;H2AX level) and late time points (24 h up to 96 h) to assess residual DSB levels, cell cycle effects and apoptosis. 
		NOTE: The protocol is not restricted to irradiation experiments or any specific cell line. It has been tested with numerous cell lines of all types, from different species and for various treatment conditions.</li>
	</ol>
	</li>
	<li>Prepare the following solutions including 10% excess volume.
	<ol>
		<li>Prepare 2 mL per sample of fixation solution composed of 4.5% paraformaldehyde (PFA) in phosphate-buffered saline (PBS). Prepare the solution fresh. Dilute PFA in PBS by heating to 80 &#176;C with slow stirring under the fume hood. Cover the flask with aluminum foil to prevent heat loss. Let the solution cool to room temperature and adjust the final volume. Pass the solution through a folded cellulose filter, grade 3hw (see the Table of Materials). 
		CAUTION: PFA fumes are toxic. Perform this step under a fume hood and dispose PFA waste appropriately.</li>
		<li>Prepare 3 mL per sample of permeabilization solution composed of 70% ethanol in ice-cold H2O. Store at -20 &#176;C.</li>
		<li>Prepare 7 mL per sample&#160;of washing solution composed of 0.5% bovine serum albumin (BSA) in PBS.</li>
		<li>Prepare 100 &#181;L per sample of 3% BSA in PBS as antibody diluent.</li>
		<li>Prepare 100-250 &#181;L per sample of DNA staining solution composed of 1 &#956;g/mL 4&#39;,6-diamidin-2-phenylindol (DAPI) in PBS.</li>
	</ol>
	</li>
	<li>Set the centrifuge for 15 mL tubes to 5 min at 200 x g and 7 &#176;C. Let the centrifuge cool down and use these settings for all centrifugation steps.</li>
</ol>
2. Sample Collection
<ol>
	<li>If processing adherent cells (e.g., U87 glioblastoma cells grown in T25 flasks with 5 mL of Dulbecco&#39;s modified Eagle&#39;s medium supplemented with 10% fetal bovine serum at 37 &#176;C and 5% carbon dioxide atmosphere), continue with steps 2.1.1. and 2.1.2. For suspension cells proceed directly to step 2.1.2.
	<ol>
		<li>Collect the medium in a centrifugation tube. Detach the cells using a routine cell culture method, which may include the use of trypsin, ethylenediaminetetraacetic acid (EDTA) or other cell detachment agents.
		<ol>
			<li>For U87 cells, prewarm PBS and trypsin/EDTA (see the Table of Materials) to 37 &#176;C, wash the cell layer with 1 mL of PBS, incubate the cells for 1-2 min with 1 mL of trypsin/EDTA and support cell detachment by tapping at the flask. Collect all washing solution and the cell suspension in the tube with the medium.</li>
		</ol>
		</li>
		<li>Centrifuge the cells, discard the medium and resuspend the cells in 1 mL of PBS.</li>
	</ol>
	</li>
	<li>Pipet the cells up and down several times to ensure a single cell suspension and transfer the cell suspension into a tube with 2 mL of fixation solution (4.5% PFA/PBS, 3% final concentration). 
	CAUTION: PFA fumes are toxic. Perform this step under a fume hood and dispose PFA waste appropriately.</li>
	<li>Incubate the cells for 10 min at room temperature.</li>
	<li>Centrifuge the cells and discard the supernatant by decantation.</li>
	<li>Loosen the cell pellet by tapping onto the tube and resuspend the cells in 3 mL of 70% ethanol. Proceed directly with the next step or store the samples at 4 &#176;C for up to several weeks.</li>
</ol>
3. Washing and Staining
<ol>
	<li>Centrifuge the cells and discard the supernatant by decantation.</li>
	<li>Loosen the cell pellet by tapping onto the tube. Resuspend the cells in 3 mL of washing solution (0.5% BSA/PBS), centrifuge and discard the supernatant.</li>
	<li>Repeat the washing step 1x with 3 mL, and then 1x with 1 mL washing solution. In the last step, discard the supernatant carefully by pipetting. Take care not to aspirate the pellet.</li>
	<li>Dilute the antibodies against &#947;H2AX, phospho-histone H3 (Ser10) and caspase-3 (see the Table of Materials) in 100 &#181;L/sample with antibody diluent (3% BSA/PBS).</li>
	<li>Loosen the cell pellet by tapping onto the tube and resuspend the cells in 100 &#181;L of the antibody solution prepared in step 3.4. Keep the samples in the dark from this step onward.</li>
	<li>Incubate the samples for 1 h at room temperature.</li>
	<li>Centrifuge the cells and discard the supernatant carefully by pipetting. Take care not to aspirate the pellet.</li>
	<li>Loosen the cell pellet by tapping onto the tube and resuspend the cells in 100-250 &#181;L of DNA staining solution (1 &#181;g/mL DAPI/PBS).
	<ol>
		<li>Use 100 &#181;L if 1-2 x105 cells are present and increase the volume for higher cell numbers (250 &#181;L for &#8805;1 x 106 cells). Proceed directly with the next step or store the samples in the dark at 4 &#176;C for up to 2 weeks. 
		NOTE: For some cell types optimizing the DAPI concentration can help to improve the separation of the cell cycle phases.</li>
	</ol>
	</li>
	<li>Pipet the samples through the cell strainer cap of a sample tube with a mesh pore size of 35 &#181;m.</li>
</ol>
4. Measurement
<ol>
	<li>Place the samples on ice, start the flow cytometer (see the Table of Materials) configured with an optical setup according to Table 1 and press the Prime button. If required, switch on the ultraviolet laser (355 nm wavelength) separately and set the power to 20 mW using the appropriate software.</li>
	<li>Open the acquisition software (see Table of Materials), log in and create a new experiment by clicking the New Experiment button on the Browser toolbar.</li>
	<li>Use the Inspector window to customize the name of the experiment and choose &#39;5 Log Decades&#39;&#160;for plot display.</li>
	<li>Click the New Specimen button in the Browser toolbar and expand the new entry by clicking the &#39;+&#39;&#160;symbol at its left side to show the first Tube. Select the respective icon and type to rename the Specimen (e.g., cell type) and the Tube (sample identifier). Click the Tube Pointer of the first Tube (arrow-like symbol at its left) to turn it green (active).</li>
	<li>Open the Parameters tab in the Cytometer window and choose the parameters according to Table 1. Delete all unnecessary parameters. 
	NOTE: the parameter names may vary depending on the custom presets (e.g., Cy3 instead of Alexa555). Make sure that the selection matches the optical filters and detectors in Table 1. The light paths of all fluorophores are fully independent in this setup and compensation of spectral overlap is not required; however, it might be necessary if another optical setup is used.</li>
	<li>Open the Worksheet window and create plots according to Figure 1. Draw 2 dot plots and 4 histograms using the corresponding toolbar buttons and click the axis labels to choose the appropriate parameters (front scatter (FSC-A) versus side scatter (SSC-A), DAPI-W versus DAPI-A and a histogram for DAPI-A and each antibody-coupled fluorophore.</li>
	<li>Attach a control sample to the cytometer and press the Run button on the instrument. Select the first tube in the Browser window of the software and click the Acquire Data button in the Acquisition Dashboard. Adjust the sample injection volume using the Low, Mid or High buttons and the fine tuning wheel on the instrument. Preferably work at Low setting, but try to acquire at least 100 events/second (see Acquisition Dashboard).</li>
	<li>Adjust the detector voltages for FSC, SSC and DAPI in the Parameters tab of the Inspector window using the dot plots in Figure 1 as a guideline. Switch to logarithmic scale for the FSC and SSC parameters if the cell population appears too dispersed on a linear scale.</li>
	<li>Press the Standby button on the cytometer and continue with the worksheet setup in the software.
	<ol>
		<li>Use the Polygon Gate tool to define the Cells population in the FSC-A versus SSC-A plot and the Rectangle Gate tool to define the SingleCells population in the DAPI-W versus DAPI-A plot. Press Ctrl + G keys to show the Population Hierarchy and click on the default gate names to rename them.</li>
		<li>Subsequently right-click on all histograms and choose Show Populations | SingleCells from the context menu.</li>
	</ol>
	</li>
	<li>Optimize the detector voltages for the antibody-coupled fluorophores to cover the full dynamic range by subsequently acquiring control and treated samples. Maximize the signal-to-noise ratio and avoid detector saturation. Make sure that the Alexa488 peak in the SingleCells population is neither truncated in the control nor the treated sample.</li>
	<li>Press the Standby button on the cytometer and optionally perform steps 4.11.1-4.11.3 in the Worksheet window of the software to get a rough estimate of treatment effects during sample acquisition.
	<ol>
		<li>Select SingleCells in the Population Hierarchy and use the Rectangle Gate tool to define the G1 population in the DAPI-W versus DAPI-A plot. Right-click at the Alexa488 histogram and choose Show Populations | G1 from the context menu.</li>
		<li>Right-click at the Alexa488 histogram and choose Create Statistics View from the context menu. Right-click at the Statistics View and choose Edit Statistics View. Go to the Statistics tab and activate the checkbox for the median of the Alexa488 signal in the G1 population (deactivate all other options).</li>
		<li>Select SingleCells in the Population Hierarchy and use the Interval Gate tool to define the subG1, M and Casp3+ populations in the DAPI-A, Alexa555-A (Cy3-A in Figure 1) and Alexa647-A histograms.</li>
	</ol>
	</li>
	<li>Press the Run button on the cytometer and measure the samples using the Acquisition Dashboard in the software. Set the stopping gate to All events or the Cells gate (if numerous small particles are present) and the number of events to record to 10,000.</li>
	<li>Click Next Tube to create a new sample, rename it in the Browser window, click Acquire Data to start the acquisition and Record Data to start recording.</li>
	<li>Select File | Export | FCS files from the menu bar to export the data. Optionally select File | Export | Experiments to save the experiment as an additional zip file to enable reimporting the experiment at a later time point. 
	NOTE: The setup of existing experiments can be easily reused with Edit | Duplicate without data.</li>
</ol>
5. Data Evaluation
<ol>
	<li>Drag and drop the &#39;.fcs&#39;&#160;files into the sample browser of the flow cytometric analysis software (see Table of Materials). Apply the gating strategy shown in Figure 2. Make sure that the gates fit to the corresponding population in all of the samples before proceeding with the next daughter gate.
	<ol>
		<li>To apply changes to all samples, select the changed gate in the sample browser, copy it by pressing Ctrl + C, select the parent gate, press Ctrl + Shift + E to select the equivalent nodes in all samples and press Ctrl + V to paste or overwrite the gate. Do not use group gates.</li>
		<li>Double-click on the first sample in the browser to open the SSC-A vs. FSC-A plot. Use the Polygon tool in the toolbar to define the Cells population (Figure 2, plot 1), excluding debris from the analysis. Make sure that the gate is wide enough towards the upper right corner to accommodate treatment-related shifts, but restrict the border facing to the lower left corner of the plot to reliably exclude the cell.</li>
		<li>Double-click on the Cells gate to open a new plot window and change the axes to DAPI-W (vertical) vs. DAPI-A (horizontal) by clicking on the axis labels. Use the Rectangle tool to define the SingleCells population (Figure 2, plot 2), excluding cell doublets or clumps from the analysis (doublets of G1 cells have the same DAPI-A intensity as G2 and M cells, but a considerably higher DAPI-W value). 
		NOTE: Varying cell counts in different samples will cause shifts in the overall DAPI-A signal strength due to equilibrium binding of DAPI to DNA. This will not affect the cell cycle analysis, but it may be necessary to adjust the right border of the single cell gate sample by sample to account for these shifts.</li>
		<li>Double-click on the SingleCells gate to open a new plot window and change the axes to show the DAPI-A histogram. Use the Bisector tool to distinguish single cells with normal DNA content (CellCycle population) from apoptotic cells with degraded DNA (subG1 population). Subsequently select the new gates in the browser and press Ctrl + R to rename them accordingly (Figure 2, plot 3).</li>
		<li>Select the CellCycle gate in the browser and choose Tool | Biology | Cell Cycle... from the menu bar to open the cell cycle modelling tool. Choose Dean-Jett-Fox25,26 in the Model section to estimate the frequency of cells in G1, S and G2/M phase (Figure 2, plot 4). Use constraints only in case of poor modelling performance (minimize the Root Mean Square deviation between model and data).</li>
		<li>Create gates for G1 (Ellipse tool), S (Polygon tool) and G2 + M (Ellipse tool) phase in the DAPI-W vs. DAPI-A plot of the CellCycle population to enable cell cycle-specific &#947;H2AX measurement (Figure 2, plot 5). Do not use the modelling tool for automatic cell cycle gating based on the DAPI-A histogram, as this can be inaccurate. 
		NOTE: It may be necessary to move the gates along the DAPI-A axis sample by sample to account for the aforementioned shifts in overall DAPI signal strength. Only move the three gates as a group and do not change the shape of individual gates to avoid bias.</li>
		<li>Use the Bisector tool to distinguish phospho-histone H3-positive (M) and -negative (M-) cells in the Alexa555-A histogram of the CellCycle population (Figure 2, plot 6). Hold Ctrl and select the gates G2 + M and M-. Press Ctrl + Shift + A to create the G2 (G2+M &#38; M-) gate.</li>
		<li>Use the Bisector tool to distinguish caspase-3-positive (Casp3+) and -negative (Casp3-) cells in the Alexa647-A histogram of the SingleCells population (Figure 2, plot 7). Set the threshold such that the average of the Casp3+ population in the untreated controls amounts to ~0.8% to assure high sensitivity and minimize assay-to-assay variations.</li>
		<li>Press Ctrl + T to open the Table Editor and configure it according to Figure 3. Drag and drop the different populations from the sample browser into the Table Editor and double-click on the rows to change the statistic, parameter and name settings. Remove the unnecessary rows that are automatically added after the drag and drop of the cell cycle modelling icon by selecting the rows and pressing Del.</li>
		<li>Choose To File, the format and destination in the Output section of the menu ribbon and click Create Table to export the data as &#39;.xlsx&#39;&#160;file.</li>
	</ol>
	</li>
	<li>Use table calculation software (see Table of Materials) for further data analysis according to Supplementary File 1 (FACS_Analysis_Template_(1).xlsx).
	<ol>
		<li>Correct the frequencies of cells in the different cell cycle phases such that their sum amounts to 100% by applying the formula X&#39;&#160;= X * 100 / &#931;(all cell cycle phases), with X&#39;: corrected value, X: raw value, to each cell cycle phase. 
		NOTE: Deviations from a sum of 100% occur due to inaccuracies in the cell cycle modelling, but are usually small (&#60;5%).</li>
		<li>Normalize the median &#947;H2AX intensities to the DNA content in the different cell cycle phases by dividing the values in S phase by 1.5 and in G2 and M phase by 2.0.</li>
		<li>To calculate the combined normalized &#947;H2AX level in the whole cell population, use the formula IA = IG1 * G1 + IS * S + IG2 * G2 + IM * M, where IA, IG1, IS, IG2, IM are normalized median &#947;H2AX intensities of all, G1, S, G2, M cells respectively and G1, S, G2, M are corrected frequency of cells in the respective cell cycle phase.</li>
		<li>For the normalized &#947;H2AX levels and the frequency of subG1 and caspase-3-positive cells, subtract the average value of the untreated controls from each sample.</li>
		<li>Calculate the mean and standard deviation of each parameter from all replicate samples and plot the results into diagrams.</li>
	</ol>
	</li>
</ol>

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N., Jett, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mathematical+analysis+of+DNA+distributions+derived+from+flow+microfluorometry.">Mathematical analysis of DNA distributions derived from flow microfluorometry.</a> Journal of Cell Biology. 60 (2), 523-527 (1974).</li><li>Fox, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+for+the+computer+analysis+of+synchronous+DNA+distributions+obtained+by+flow+cytometry.">A model for the computer analysis of synchronous DNA distributions obtained by flow cytometry.</a> Cytometry. 1 (1), 71-77 (1980).</li><li>Costes, S. V., Boissi&#232;re, A., Ravani, S., Romano, R., Parvin, B., Barcellos-Hoff, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+features+that+discriminate+between+foci+induced+by+high-+and+low-LET+radiation+in+human+fibroblasts.">Imaging features that discriminate between foci induced by high- and low-LET radiation in human fibroblasts.</a> Radiation Research. 165 (5), 505-515 (2006).</li><li>Meyer, B., Voss, K. -. O., Tobias, F., Jakob, B., Durante, M., Taucher-Scholz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clustered+DNA+damage+induces+pan-nuclear+H2AX+phosphorylation+mediated+by+ATM+and+DNA-PK.">Clustered DNA damage induces pan-nuclear H2AX phosphorylation mediated by ATM and DNA-PK.</a> Nucleic Acids Research. 41 (12), 6109-6118 (2013).</li><li>Lopez Perez, R., 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=Superresolution+light+microscopy+shows+nanostructure+of+carbon+ion+radiation-induced+DNA+double-strand+break+repair+foci.">Superresolution light microscopy shows nanostructure of carbon ion radiation-induced DNA double-strand break repair foci.</a> FASEB. 30 (8), 2767-2776 (2016).</li><li>Schipler, A., Iliakis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+double-strand-break+complexity+levels+and+their+possible+contributions+to+the+probability+for+error-prone+processing+and+repair+pathway+choice.">DNA double-strand-break complexity levels and their possible contributions to the probability for error-prone processing and repair pathway choice.</a> Nucleic Acids Research. 41 (16), 7589-7605 (2013).</li><li>Stenerlow, B., Hoglund, E., Carlsson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+fragmentation+by+charged+particle+tracks.">DNA fragmentation by charged particle tracks.</a> Advances in Space Research. 30 (4), 859-863 (2002).</li><li>Friedland, W., 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=Comprehensive+track-structure+based+evaluation+of+DNA+damage+by+light+ions+from+radiotherapy-relevant+energies+down+to+stopping.">Comprehensive track-structure based evaluation of DNA damage by light ions from radiotherapy-relevant energies down to stopping.</a> Scientific Reports. 7, 45161 (2017).</li><li>Pang, D., Chasovskikh, S., Rodgers, J. E., Dritschilo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+DNA+Fragments+Are+a+Hallmark+of+Heavy+Charged-Particle+Irradiation+and+May+Underlie+Their+Greater+Therapeutic+Efficacy.">Short DNA Fragments Are a Hallmark of Heavy Charged-Particle Irradiation and May Underlie Their Greater Therapeutic Efficacy.</a> Frontiers in Oncology. 6, 130 (2016).</li><li>B&#246;cker, W., Iliakis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computational+Methods+for+analysis+of+foci:+validation+for+radiation-induced+gamma-H2AX+foci+in+human+cells.">Computational Methods for analysis of foci: validation for radiation-induced gamma-H2AX foci in human cells.</a> Radiation Research. 165 (1), 113-124 (2006).</li><li>L&#246;brich, 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=&#947;H2AX+foci+analysis+for+monitoring+DNA+double-strand+break+repair:+Strengths,+limitations+and+optimization.">&#947;H2AX foci analysis for monitoring DNA double-strand break repair: Strengths, limitations and optimization.</a> Cell Cycle. 9 (4), 662-669 (2010).</li></ol>

The authors have nothing to disclose.

The featured method is easy to use and offers a fast, accurate and reproducible measurement of the DNA damage response including double-strand break (DSB) induction and repair, cell cycle effects and apoptotic cell death. The combination of these endpoints provides a more complete picture of their interrelations than individual assays. The method can be widely applied as a comprehensive genotoxicity assay in the fields of radiation biology, therapy and protection, and more generally in oncology (e.g., for environmental risk factor assessment, drug screening and evaluation of genetic instability in tumor cells).
The most critical step in this protocol is the fixation of the cells. To obtain a clean sample with a clearly defined cell population and optimal resolution of different cell cycle phases, it is important to give adherent cells enough time to detach from the culture vessel and form a completely round shape. The cells should be transferred into the fixation solution as a single cell suspension without cell clumps. To separate them, the cells must be pipetted up and down or passed through a fine needle several times in difficult cases. The fixation time should be kept constant between all samples and should not exceed 20 min including the time for centrifugation before resuspension of the cells in 70% ethanol. Prolonged fixation will lead to loss of accessible epitopes for antibody binding and will decrease the signal strength and sensitivity of the method.
The main limitation of the technique is that it does not provide information on the quality of the &#947;H2AX foci other than the intensity in the whole nucleus. Particularly large &#947;H2AX foci as well as the occurrence of a pan-nuclear &#947;H2AX staining have been linked to clustered DSBs1,27,28,29, which are highly complex lesions that are very difficult to repair30. Such clustered lesions seem to be characteristic for particle radiation such as clinically applied carbon ion radiation and may be the reason for the superior biological effectiveness of heavy ion radiation compared to photons31,32,33. Analysis of the &#947;H2AX foci size may therefore be of interest as an indicator of the damage complexity. Although it is possible to use the present protocol with an image stream cytometer to visualize the &#947;H2AX foci, the achievable resolution cannot compete with a classical microscopic approach. However, we have successfully prepared microscopic slides from the remaining samples after flow cytometric measurement (Supplementary Figure 1) and evaluated several features including &#947;H2AX foci count, size and pan-nuclear intensity using a semi-automated imaging and analysis system. To prepare the samples for microscopy, 30-50 &#181;L of the stained cells were spread over the surface of a 24 mm x 24 mm cover glass, air dried overnight at room temperature in the dark and then embedded with mounting medium on a glass slide.
In comparison with microscopic evaluation of DSB levels by &#947;H2AX foci counting, the flow cytometric measurement is superior in throughput, sampling rate, dynamic range and accuracy. The dynamic range of microscopic foci counting is limited by the optical resolution, leading to the inability to distinguish overlapping foci29,34. In practice, we have observed a linear relationship between radiation dose and &#947;H2AX foci numbers below 2 Gy, and a strong saturation effect above 2 Gy in U87 glioblastoma cells1. In contrast, the intensity of the &#947;H2AX signal measured by flow cytometry increased linearly with dose for the whole dose range tested (0-8 Gy).
The accuracy of microscopic foci counting is limited by the inability to distinguish between different cell cycle phases without additional stainings35. The number of DSBs induced in a cell is not only dependent on the radiation dose, but also on the DNA content, and hence the cell cycle stage. G2 cells for example have twice as much DNA as G1 cells and the average number of DSBs induced at a certain radiation dose is twice as high for G2 cells compared to G1. This is clearly reflected in the &#947;H2AX signal intensity of G2 versus G1 cells at early time points after radiation measured by flow cytometry. Therefore, it is important to evaluate DSB levels in a cell cycle-specific manner to obtain accurate results. A pooled analysis of cells in different cell cycle phases, like usual in microscopic approaches, can be biased by shifts in the cell cycle distribution particularly due to radiation-induced G2 arrest. To account for this effect and to compare DSB repair characteristics between different cell cycle stages, the &#947;H2AX levels can easily be normalized to the DNA content in the flow cytometry method as indicated in the protocol.
Extensions of the present protocol are possible with relatively little extra effort. Additional antibodies with compatible fluorophores labels can be included in step 3.4 without the need for any extra steps during sample preparation. If a more detailed analysis of apoptosis is desired, caspase-7, -9 and -8 antibodies could be included. As another extension, we have recently included a live and dead cell dye, a troponin T antibody and counting beads in the protocol to specifically analyze cardiomyocytes co-cultured with fibroblasts after irradiation. The addition of the live/dead cell stain and the counting beads expands the capability of the method to include fibroblast proliferation and non-apoptotic cell death of the cardiomyocytes as further endpoints that provide useful additional information on the cell fate after genotoxic stress. The extended protocol is available upon request.

The goal of oncology is to kill or to inactivate cancer cells without harming normal cells. Many therapies either directly or indirectly induce genotoxic stress in cancer cells, but also to some extend in normal cells. Chemotherapy or targeted drugs are often combined with radiotherapy to enhance the radiosensitivity of the irradiated tumor1,2,3,4,5, which allows for a reduction of the radiation dose to minimize normal tissue damage.
Ionizing radiation and other genotoxic agents induce different kinds of DNA damage, including base modifications, strand crosslinks and single- or double-strand breaks. DNA double-strand breaks (DSBs) are the most serious DNA lesions and their induction is key to the cell killing effect of ionizing radiation and various cytostatic drugs in radiochemotherapy. DSBs do not only harm the integrity of the genome, but also promote the formation of mutations6,7. Therefore, different DSB repair pathways, and mechanisms to eliminate irreparably damaged cells like apoptosis have developed during evolution. The entire DNA damage response (DDR) is regulated by a complex network of signaling pathways that reach from DNA damage recognition and cell cycle arrest to allow for DNA repair, to programmed cell death or inactivation in case of repair failure8.
The presented flow cytometric method has been developed to investigate the DDR after genotoxic stress in one comprehensive assay that covers DSB induction and repair, as well as consequences of repair failure. It combines the measurement of the widely applied DSB marker &#947;H2AX with analysis of the cell cycle and induction of apoptosis, using classical subG1 analysis and more specific evaluation of caspase-3 activation.
The combination of these endpoints in one assay not only reduces time, labor and cost expenses, but also enables cell cycle-specific measurement of DSB induction and repair, as well as caspase-3 activation. Such analyses would not be possible with independently conducted assays, but they are highly relevant for a comprehensive understanding of the DNA damage response after genotoxic stress. Many anti-cancer drugs, such as cytostatic compounds, are directed against dividing cells and their efficiency is strongly dependent on the cell cycle stage. The availability of different DSB repair processes is also dependent on the cell cycle stage and pathway choice which is critical for the repair accuracy, and in turn determines the fate of the cell9,10,11,12. In addition, cell cycle-specific measurement of DSB levels is more accurate than pooled analysis, because DSB levels are not only dependent on the dose of a genotoxic compound or radiation, but also on the DNA content of the cell.
The method has been used to compare the efficacy of different radiotherapies to overcome resistance mechanisms in glioblastoma13 and to dissect the interplay between ionizing radiation and targeted drugs in osteosarcoma14,15 and atypical teratoid rhabdoid cancers16. Additionally, the described method has been widely used to analyze side effects of radio- and chemotherapy on mesenchymal stem cells17,18,19,20,21,22,23,24, which are essential for the repair of treatment-induced normal tissue damage and have a potential application in regenerative medicine.

<table><tbody><tr><td>1,000 &amp;micro;L filter tips</td><td>Nerbe plus</td><td>07-693-8300</td><td></td></tr><tr><td>100-1,000 &amp;micro;L pipette</td><td>Eppendorf</td><td>3123000063</td><td></td></tr><tr><td>12 mm x 75 mm Tubes with Cell Strainer Cap, 35 &amp;micro;m mesh pore size</td><td>BD Falcon</td><td>352235</td><td></td></tr><tr><td>15 mL tubes</td><td>BD Falcon</td><td>352096</td><td></td></tr><tr><td>200 &amp;micro;L filter tips</td><td>Nerbe plus</td><td>07-662-8300</td><td></td></tr><tr><td>20-200 &amp;micro;L pipette</td><td>Eppendorf</td><td>3123000055</td><td></td></tr><tr><td>4&amp;rsquo;,6-Diamidin-2-phenylindol (DAPI)</td><td>Sigma-Aldrich</td><td>D9542</td><td>Dissolve in water at 200 &amp;micro;g/mL and store aliquots at -20 &amp;deg;C</td></tr><tr><td>Alexa Fluor 488 anti-H2A.X Phospho (Ser139) Antibody, RRID: AB_2248011</td><td>BioLegend</td><td>613406</td><td>Dilute 1:20</td></tr><tr><td>Alexa Fluor 647 Rabbit Anti-Active Caspase-3 Antibody, AB_1727414</td><td>BD Pharmingen</td><td>560626</td><td>Dilute 1:20</td></tr><tr><td>BD FACSClean solution</td><td>BD Biosciences</td><td>340345</td><td>For cytometer cleaning routine after measurement</td></tr><tr><td>BD FACSRinse solution</td><td>BD Biosciences</td><td>340346</td><td>For cytometer cleaning routine after measurement</td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline (PBS)</td><td>Biochrom</td><td>L 182</td><td>Dissolve in water to 1x concentration</td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium with stable glutamin</td><td>Biochrom</td><td>FG 0415</td><td>Routine cell culture material for the example cell line used in the protocol</td></tr><tr><td>Ethanol absolute</td><td>VWR</td><td>20821.330</td><td></td></tr><tr><td>Excel software</td><td>Microsoft</td><td></td><td></td></tr><tr><td>FBS Superior (fetal bovine serum)</td><td>Biochrom</td><td>S 0615</td><td>Routine cell culture material for the example cell line used in the protocol</td></tr><tr><td>FlowJo v10 software</td><td>LLC</td><td>online order</td><td></td></tr><tr><td>Fluoromount-G</td><td>SouthernBiotech</td><td>0100-01</td><td>Embedding medium for optional preparation of microscopic slides from stained samples</td></tr><tr><td>Folded cellulose filters, grade 3hw</td><td>NeoLab</td><td>11416</td><td></td></tr><tr><td>LSRII or LSRFortessa cytometer</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>MG132</td><td>Calbiochem</td><td>474787</td><td>optional drug for apoptosis positive control</td></tr><tr><td>Multifuge 3SR+</td><td>Heraeus</td><td></td><td></td></tr><tr><td>Paraformaldehyde</td><td>AppliChem</td><td>A3813</td><td>Prepare 4.5% solution fresh. Dilute in PBS by heating to 80 &amp;deg;C with slow stirring under the fume hood. Cover the flask with aluminium foil to prevent heat loss. Let the solution cool to room temperature and adjust the final volume. Pass the solution through a cellulose filter.</td></tr><tr><td>Phospho-Histone H3 (Ser10) (D2C8) XP Rabbit mAb (Alexa Fluor&amp;reg; 555 Conjugate) RRID: AB_10694639</td><td>Cell Signaling Technology</td><td>#3475</td><td>Dilute 3:200</td></tr><tr><td>PIPETBOY acu 2</td><td>Integra Biosciences</td><td>155 016</td><td></td></tr><tr><td>Serological pipettes, 10 mL</td><td>Corning</td><td>4488</td><td></td></tr><tr><td>Serological pipettes, 25 mL</td><td>Corning</td><td>4489</td><td></td></tr><tr><td>Serological pipettes, 5 mL</td><td>Corning</td><td>4487</td><td></td></tr><tr><td>SuperKillerTRAIL (modified TNF-related apoptosis-inducing ligand)</td><td>Biomol</td><td>AG-40T-0002-C020</td><td>optional drug for apoptosis positive control</td></tr><tr><td>T25 cell culture flasks</td><td>Greiner bio-one</td><td>690160</td><td>Routine cell culture material for the example cell line used in the protocol</td></tr><tr><td>Trypsin/EDTA</td><td>PAN Biotech</td><td>P10-025500</td><td>Routine cell culture material for the example cell line used in the protocol</td></tr><tr><td>U87 MG glioblastoma cells</td><td>ATCC</td><td>ATCC-HTB-14</td><td>Example cell line used in the protocol</td></tr></tbody></table>

cell cycle-specific measurement of &#947;h2ax and apoptosis after genotoxic stress by flow cytometry

The presented method or slightly modified versions have been devised to study specific treatment responses and side effects of various anti-cancer treatments as used in clinical oncology. It enables a quantitative and longitudinal analysis of the DNA damage response after genotoxic stress, as induced by radiotherapy and a multitude of anti-cancer drugs. The method covers all stages of the DNA damage response, providing endpoints for induction and repair of DNA double-strand breaks (DSBs), cell cycle arrest and cell death by apoptosis in case of repair failure. Combining these measurements provides information about cell cycle-dependent treatment effects and thus allows an in-depth study of the interplay between cellular proliferation and coping mechanisms against DNA damage. As the effect of many cancer therapeutics including chemotherapeutic agents and ionizing radiation is limited to or strongly varies according to specific cell cycle phases, correlative analyses rely on a robust and feasible method to assess the treatment effects on the DNA in a cell cycle-specific manner. This is not possible with single-endpoint assays and an important advantage of the presented method. The method is not restricted to any particular cell line and has been thoroughly tested in a multitude of tumor and normal tissue cell lines. It can be widely applied as a comprehensive genotoxicity assay in many fields of oncology besides radio-oncology, including environmental risk factor assessment, drug screening and evaluation of genetic instability in tumor cells.

Human U87 or LN229 glioblastoma cells were irradiated with 4 Gy of photon or carbon ion radiation. Cell cycle-specific &#947;H2AX levels and apoptosis were measured at different time points up to 48 h after irradiation using the flow cytometric method presented here (Figure 3). In both cell lines, carbon ions induced higher &#947;H2AX peak levels that declined slower and remained significantly elevated at 24 to 48 h compared to photon radiation at the same physical dose (Figure 4A). This indicated that carbon ions induced higher peak levels of DNA double-strand breaks (DSBs) than photons that were repaired less efficiently. For both radiation types, the &#947;H2AX levels were highest in G1 cells, probably because DSB repair is limited to the pathway of non-homologous end-joining at this stage of the cell cycle.
In line with the higher DSB induction rate and slower repair kinetics, carbon ions induced a stronger and longer-lasting cell cycle arrest in G2 phase (Figure 3B) and a higher rate of apoptosis than photons (Figure 4C). Carbon ion radiation may therefore help to overcome radioresistance mechanisms observed in glioblastoma upon classical radiotherapy with photons (see Lopez Perez, et al.1 for detailed discussion).
The results shown in Figure 4 are examples of an optimal outcome of this method, showing clear differences between untreated and irradiated cells and statistically significant differential effects between different treatments. In cases where induction of DSBs and apoptosis is less clear, it is important to include positive controls. As shown here, cells fixed 1 h after photon irradiation are good positive controls for &#947;H2AX induction. Photon radiation is also known to efficiently induce apoptosis in lymphocytes, which makes them useful as positive controls for apoptosis 2-4 days after irradiation. Another possibility is to use drugs for apoptosis induction, such as the proteasome inhibitor MG132 or a death receptor ligand (Figure 5).
Another important aspect for an optimal outcome of the assay is the quality of the cell cycle profiles. In Figure 4B the G1 and G2 peaks were narrow and clearly separated, making it easy to apply cell cycle modelling and to define the gates for cell-cycle-specific measurement of &#947;H2AX. Depending on the cell type and the strength of the treatment effect (Figure 6A,B), the resolution of the different cell cycle phases can be significantly worse. In some cases, the DAPI concentration has a big impact on the quality of the cell cycle profile and needs to be adjusted (Figure 6C). In cases where no clear G1 peak is detectable due to the treatment, the cell cycle modelling tool cannot be applied accurately. It is advisable then to only use manual gating in the DAPI-A versus DAPI-W plot based on controls with a well-defined cell cycle profile, as described in the protocol.
The analysis of a cell cycle profile without a clear G1 peak can be further complicated if the treatment leads to low cell numbers that result in large shifts in the overall DAPI signal strength. In this situation, it can be difficult to judge, if a peak is the G1 or the G2 peak. To avoid this problem, the cells can be counted with a Neubauer chamber or by other means after the last washing step (step 3.3) and the cell numbers can be adjusted. The peak positions in the treated cells will then match with the controls.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59968/59968fig1.jpg" /> 
Figure 1: Worksheet setup for sample acquisition. Plots and gates for signal display in the Worksheet window of the flow cytometer software (see the Table of Materials). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59968/59968fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59968/59968fig2v2.jpg" /> 
Figure 2: Gating strategy for data evaluation. Population tree and diagrams showing how the gates are set to define the different sub-populations in the flow cytometric analysis software. DJF refers to the cell cycle modelling tool using the Dean-Jett-Fox method25,26. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59968/59968fig2v2large.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/59968/59968fig3.jpg" /> 
Figure 3: Raw data export table. Setup of the &#39;Table Editor&#39;&#160;for raw data export in the flow cytometric analysis software. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59968/59968fig3large.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/59968/59968fig4v2.jpg" /> 
Figure 4: DNA damage response of human U87 and LN229 glioblastoma cells after irradiation with 4 Gy of photon versus carbon ion radiation. (A) DSB induction and repair measured by &#947;H2AX levels. The median fluorescence intensity was normalized to the relative DNA content in each cell cycle phase (G1 = 1.0, S = 1.5, G2 = 2.0) and control levels were subtracted. Symbols indicate mean and error bars indicate SD values. Solid lines represent fits according to the function I(t) = (p1*t&#178; + p2*t) / (t&#178; + q1*t + q2) | t &#8805; 0, p1 &#60; 0, p1, q1, q2 &#62; 0. (B) Cell cycle distribution (mean and SD) and representative histograms of the DNA content marker DAPI at 24 h after photon or carbon ion irradiation. (C) Apoptosis induction, as measured by caspase-3 and subG1 positive cells (mean and SD). *P &#60; 0.05, **P &#60; 0.01, ***P &#60; 0.001 (two-sided Student&#39;s t test). This figure has been modified from Lopez, et al.1 with permission. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59968/59968fig4v2large.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/59968/59968fig5.jpg" /> 
Figure 5: Drug-mediated apoptosis-induction in U87 glioblastoma cells. U87 cells were treated with 5 ng/mL of a modified TNF-related apoptosis-inducing ligand (see Table of Materials) plus 2.5 &#181;M MG132 for 20 h before fixation to validate the apoptosis measurement by active caspase-3 and subG1 analysis. (A) Histogram of the caspase-3 signal of treated versus untreated cells. (B) DAPI histogram of treated cells with a subG1 population, indicating DNA degradation. The histogram of caspase-3-positive events is overlaid in red, demonstrating that most of the apoptotic cells had not yet degraded their DNA at this time point. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59968/59968fig5large.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/59968/59968fig6.jpg" /> 
Figure 6: Factors influencing the quality of cell cycle profiles. Too harsh treatments complicate cell cycle analysis. (A) No G1 peak is detectable 48 h after treatment of LLC cells (Lewis lung carcinoma) with 20 Gy of photon radiation or (B) 96 h after treatment of HS68 fibroblasts with 100 mJ of UV radiation. (C) DAPI concentration in different cell lines: In HS68 fibroblasts (upper panel) the G2/M peak (see arrows) was equally well-separated from the G1 peak for DAPI concentrations between 0.01 and 1.0 &#181;g/mL. In contrast, DAPI concentrations below 0.75 &#181;g/mL resulted in poor separation and shape definition of the G2/M peak (see arrows) in MRC-5 fibroblasts (lower panel). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59968/59968fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Parameter</td>
			<td>Significance</td>
			<td colspan="2">Excitation laser</td>
			<td colspan="2">Detector</td>
			<td colspan="4">Signal</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>&#955; (nm)</td>
			<td>P (mW)</td>
			<td>Filter</td>
			<td>U (V)</td>
			<td>log</td>
			<td>-A</td>
			<td>-H</td>
			<td>-W</td>
		</tr>
		<tr>
			<td>FSC</td>
			<td>forward scatter (size)</td>
			<td>488</td>
			<td>100</td>
			<td>488/10</td>
			<td>230</td>
			<td></td>
			<td>&#9679;</td>
			<td>&#9679;</td>
			<td></td>
		</tr>
		<tr>
			<td>SSC</td>
			<td>side scatter (granularity)</td>
			<td>488</td>
			<td>100</td>
			<td>488/10</td>
			<td>210</td>
			<td></td>
			<td>&#9679;</td>
			<td>&#9679;</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa488</td>
			<td>&#947;H2AX (double-strand breaks)</td>
			<td>488</td>
			<td>100</td>
			<td>525/50</td>
			<td>275</td>
			<td>&#9679;</td>
			<td>&#9679;</td>
			<td>&#9679;</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa555</td>
			<td>phospho-histone H3 (M phase)</td>
			<td>561</td>
			<td>150</td>
			<td>586/15</td>
			<td>360</td>
			<td>&#9679;</td>
			<td>&#9679;</td>
			<td>&#9679;</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa647</td>
			<td>active caspase-3 (apoptosis)</td>
			<td>640</td>
			<td>40</td>
			<td>670/14</td>
			<td>410</td>
			<td>&#9679;</td>
			<td>&#9679;</td>
			<td>&#9679;</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>DNA (cell cycle, apoptosis)</td>
			<td>350</td>
			<td>20</td>
			<td>450/50</td>
			<td>350</td>
			<td></td>
			<td>&#9679;</td>
			<td>&#9679;</td>
			<td>&#9679;</td>
		</tr>
	</tbody>
</table>
Table 1: Typical flow cytometer configuration. &#955; = wavelength of the excitation laser, U = detector voltage, log = logarithmic scale (otherwise linear), -A = signal area, -H = maximum signal height, -W = signal width (duration), FSC = front scatter, SSC = side scatter. Optical filter specifications are given as central wavelength/bandwidth in nanometers. Settings may vary for different flow cytometers and detector voltages need to be adjusted for different cell lines. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59968/table1.xlsx" target="_blank">Please click here to view this table (Right click to download).</a>
<img alt="Figure 1" src="/files/ftp_upload/59968/59968supfig1.jpg" /> 
Supplementary Figure 1: Microscopic images of &#947;H2AX foci. U87 cells were irradiated with different doses of photon radiation, fixed and stained after 30 min according to the presented protocol and prepared for microscopy as described in the discussion. At 2 Gy, individual foci could be easily distinguished, while at 8 Gy foci counting was biased due to considerable overlap. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59968/59968supfig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary File 1:&#160;<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59968/FACS_Analysis_Template_(1).xlsx" target="_blank">Please click here to download this file.</a>

Watch this Scientific Journal Video about Cell Cycle-specific Measurement of Î³H2AX and Apoptosis After Genotoxic Stress by Flow Cytometry at JoVE.com

Cell Cycle-specific Measurement of &#947;H2AX and Apoptosis After Genotoxic Stress by Flow Cytometry

The presented method combines the quantitative analysis of DNA double-strand breaks (DSBs), cell cycle distribution and apoptosis to enable cell cycle-specific evaluation of DSB induction and repair as well as the consequences of repair failure.

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

cell-cycle-specific-measurement-h2ax-apoptosis-after-genotoxic-stress

Nanyang Technological University

Research

JoVE Journal

Cancer Research

13.3K Views.  German Cancer Research Center. The presented method combines the quantitative analysis of DNA double-strand breaks (DSBs), cell cycle distribution and apoptosis to enable cell cycle-specific evaluation of DSB induction and repair as well as the consequences of repair failure.

Video: Cell Cycle-specific Measurement of γH2AX and Apoptosis After Genotoxic Stress by Flow Cytometry

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.

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 ...

An Automated Microscopic Scoring Method for the &#947;-H2AX Foci Assay in Human Peripheral Blood Lymphocytes

Ionizing radiation is a potent inducer of DNA damage and a well-documented carcinogen. Biological dosimetry comprises the detection of biological effects induced by exposure to ionizing radiation to make an individual dose assessment. This is pertinent in the framework of radiation emergencies, where health assessments and planning of clinical treatment for exposed victims are critical. Since DNA double strand breaks (DSB) are considered to be the most lethal form of radiation-induced DNA damage, this protocol presents a method to detect DNA DSB in blood samples. The methodology is based on the detection of a fluorescent labelled phosphorylated DNA repair protein, namely, &#947;-H2AX. The use of an automated microscopy platform to score the number of &#947;-H2AX foci per cell allows a standardized analysis with a significant decrease in the turn-around time. Therefore, the &#947;-H2AX assay has the potential to be one of the fastest and sensitive assays for biological dosimetry. In this protocol, whole blood samples from healthy adult volunteers will be processed and irradiated in vitro in order to illustrate the usage of the automated and sensitive &#947;-H2AX foci assay for biodosimetry applications. An automated slide scanning system and analysis platform with an integrated fluorescence microscope is used, which allows the fast, automatic scoring of DNA DSB with a reduced degree of bias.

This protocol presents the slide preparation and automated scoring of the &#947;-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.

Ionizing radiation is a potent inducer of DNA damage and a well-documented carcinogen. Biological dosimetry comprises the detection of biological effects ...

Flow Cytometric Analysis of Apoptotic Biomarkers in Actinomycin D-Treated SiHa Cervical Cancer Cells

Apoptosis biomarkers were investigated in actinomycin D-treated SiHa cervical cancer cells using a benchtop flow cytometer. Early biomarkers (Annexin V and mitochondrial membrane potential) and late biomarkers (caspases 3 and 7, and DNA damage) of apoptosis were measured in experimental and control cultures. Cultures were incubated for 24 hours in a humidified incubator at 37 &#176;C with 5% CO2. The cells were then detached using trypsin and enumerated using a flow cytometric cell count assay. Cells were further analyzed for apoptosis using an Annexin V assay, a mitochondrial electrochemical transmembrane potential assay, a caspase 3/7 assay, and a DNA damage assay. This article provides an overview of apoptosis and traditional flow cytometry, and elaborates flow cytometric protocols for processing and analyzing SiHa cells. The results describe positive, negative, and sub-optimal experimental data. Also discussed are interpretation and caveats in performing flow cytometric analysis of apoptosis using this analytical platform. Flow cytometric analysis provides an accurate measurement of early and late biomarkers for apoptosis.

Apoptosis can be characterized by flow cytometric analysis of early and late apoptotic biomarkers. The cervical cancer cell line, SiHa, was analyzed for apoptosis biomarkers after treatment with Actinomycin D using a benchtop flow cytometer.

Apoptosis biomarkers were investigated in actinomycin D-treated SiHa cervical cancer cells using a benchtop flow cytometer. Early biomarkers (Annexin V ...

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

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

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

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

Genetics

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

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

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

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

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

Biology

Quantitation of &gamma;H2AX Foci in Tissue Samples

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

Quantitation of &#947;H2AX Foci in Tissue Samples

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

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

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

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

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

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

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

Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell synchronization can be difficult for many cell systems. Standard practice is to block cell cycle progression at a specific stage and then release the accumulated cells producing a wave of cells progressing through the cycle in unison. However, some cell types find this block toxic resulting in abnormal cell cycling, or even mass death. Bromodeoxyuridine (BrdU) uptake can be used to track the cell cycle stage of individual cells. Cells incorporate this synthetic thymidine analog, while synthesizing new DNA during S phase. By providing BrdU for a brief period it is possible to mark a pool of cells that were in S phase while the BrdU was present. These cells can then be tracked through the remainder of the cell cycle and into the next round of replication, permitting the duration of the cell cycle phases to be determined without the need to induce a potentially toxic cell cycle block. It is also possible to determine and correlate the expression of both internal and external proteins during subsequent stages of the cell cycle. These can be used to further refine the assignment of cell cycle stage or assess effects on other cellular functions such as checkpoint activation or cell death.

This protocol describes the use of bromodeoxyuridine (BrdU) uptake to permit the temporal tracking of cells that were in S phase at a specific point in time. Addition of DNA dyes and antibody labeling facilitates detailed analysis of the fate of the S phase cells at later times.

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell ...

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high radiation doses. Addressing questions related to environmental, occupational and public health safety in a proper and scientifically justified manner heavily relies on the ability to accurately measure the biological effects of low dose pollutants, such as ionizing radiation and chemical substances. DNA damage and repair are the most important early indicators of health risks due to their potential long term consequences, such as cancer. Here we describe a protocol to study the effect of chronic in vivo exposure to low doses of &#947;- and &#946;-radiation on DNA damage and repair in mouse spleen cells. Using a commonly accepted marker of DNA double-strand breaks, phosphorylated histone H2AX called &#947;H2AX, we demonstrate how it can be used to evaluate not only the levels of DNA damage, but also changes in the DNA repair capacity potentially produced by low dose in vivo exposures. Flow cytometry allows fast, accurate and reliable measurement of immunofluorescently labeled &#947;H2AX in a large number of samples. DNA double-strand break repair can be evaluated by exposing extracted splenocytes to a challenging dose of 2 Gy to produce a sufficient number of DNA breaks to trigger repair and by measuring the induced (1 hr post-irradiation) and residual DNA damage (24 hrs post-irradiation). Residual DNA damage would be indicative of incomplete repair and the risk of long-term genomic instability and cancer. Combined with other assays and end-points that can easily be measured in such in vivo studies (e.g., chromosomal aberrations, micronuclei frequencies in bone marrow reticulocytes, gene expression, etc.), this approach allows an accurate and contextual evaluation of the biological effects of low level stressors.

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

A protocol to evaluate changes in DNA damage levels and DNA repair capacity that may be induced by chronic in vivo low dose irradiation in mouse spleen lymphocytes, by measuring phosphorylated histone H2AX, a marker of DNA double-strand breaks, using flow cytometry is presented.

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high ...

Techniques to Induce and Quantify Cellular Senescence

In response to cellular stress or damage, proliferating cells can induce a specific program that initiates a state of long-term cell-cycle arrest, termed cellular senescence. Accumulation of senescent cells occurs with organismal aging and through continual culturing in vitro. Senescent cells influence many biological processes, including embryonic development, tissue repair and regeneration, tumor suppression, and aging. Hallmarks of senescent cells include, but are not limited to, increased senescence-associated &#946;-galactosidase activity (SA-&#946;-gal); p16INK4A, p53, and p21 levels; higher levels of DNA damage, including &#947;-H2AX; the formation of Senescence-associated Heterochromatin Foci (SAHF); and the acquisition of a Senescence-associated Secretory Phenotype (SASP), a phenomenon characterized by the secretion of a number of pro-inflammatory cytokines and signaling molecules. Here, we describe protocols for both replicative and DNA damage-induced senescence in cultured cells. In addition, we highlight techniques to monitor the senescent phenotype using several senescence-associated markers, including SA-&#946;-gal, &#947;-H2AX and SAHF staining, and to quantify protein and mRNA levels of cell cycle regulators and SASP factors. These methods can be applied to the assessment of senescence in various models and tissues.

Cellular senescence, the irreversible state of cell-cycle arrest, can be induced by various cellular stresses. Here, we describe protocols to induce cellular senescence and methods to assess markers of senescence.

In response to cellular stress or damage, proliferating cells can induce a specific program that initiates a state of long-term cell-cycle arrest, termed ...

Cell Cycle-specific Measurement of γH2AX and Apoptosis After Genotoxic Stress by Flow Cytometry

Summary

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Chapters in this video

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

An Automated Microscopic Scoring Method for the γ-H2AX Foci Assay in Human Peripheral Blood Lymphocytes

Flow Cytometric Analysis of Apoptotic Biomarkers in Actinomycin D-Treated SiHa Cervical Cancer Cells

Dual Immunofluorescence of γH2AX and 53BP1 in Human Peripheral Lymphocytes

Quantification of γH2AX Foci in Response to Ionising Radiation

Quantitation of γH2AX Foci in Tissue Samples

Evaluation of the Spatial Distribution of γH2AX following Ionizing Radiation

Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

Techniques to Induce and Quantify Cellular Senescence