The authors would like to thank the study participants for their blood donations, as well as Nurse V. Prince for blood sample collection. The financial assistance of the South African National Research Foundation (NRF) towards this research is hereby acknowledged. Opinions expressed, and conclusions arrived at, are those of the authors and are not necessarily to be attributed to the NRF. This work was financially supported by the International Atomic Energy Agency (IAEA), through a Technical Cooperation Project (number: URU6042) to support W. Mart&#237;nez-L&#243;pez, as well as the Coordinated Research Project E35010 (contract number: 22248).&#160;

This study was approved by the Biomedical Research Ethics Committee of The University of Western Cape Ethics Committee - Code BM18/6/12.
1. Preparation of Solutions48
<ol>
	<li>Cell fixation solution (3% PFA in PBS)
	<ol>
		<li>Wear the appropriate personal protective equipment (PPE) and working in a fume hood, add 20 g of paraformaldehyde to 200 mL of distilled H2O. Heat the mixture to 60 &#176;C to aid dissolution.</li>
		<li>Once the PFA is dissolved, add 40 drops of 5&#160;N&#160;NaOH carefully over 30 minutes and to cool to room temperature. Adjust to pH 7 using 1 M HCl and make up to a final volume of 250 mL with distilled H2O (aliquots can be stored at -20 &#176;C for 1 year). Add 25&#160;mL from this solution to 41.7 mL PBS to afford a 3% PFA solution.</li>
	</ol>
	</li>
	<li>1% Bovine Serum Albumin (BSA) solution: Add 10 g of BSA to 1000 mL of PBS and mix until dissolved. Store at 4 &#176;C until use (maximum 72 hours).</li>
	<li>Triton-X Solution in a concentration of 1:500: Add 1 &#181;L of Triton-X to 500 &#181;L of PBS, store at 4 &#176;C until use (maximum 24 hours).</li>
	<li>Antibody Solutions
	<ol>
		<li>Primary antibody - Anti-&#947;-H2AX in concentration of 1:500: Add 1 &#181;L of anti-&#947;-H2AX to 500 &#181;L of 1% BSA solution, store at 4 &#176;C until use (maximum 24 hours).</li>
		<li>Secondary antibody - DAM-TRITC in concentration of 1:1000: Add 1 &#181;L of DAM-TRITC to 1000 &#181;L of 1% BSA solution, store at 4 &#176;C until use (maximum 24 hours).</li>
	</ol>
	</li>
	<li>Complete Roswell Park Memorial Institute (cRPMI) Media
	<ol>
		<li>Add filtered Fetal Bovine Serum (FBS) and Penicillin-Streptomycin to RPMI 1640 media in a sterile bottle to give a final concentration of 10% FBS and 1% Penicillin-Streptomycin.</li>
	</ol>
	</li>
</ol>
2. Preparation of Samples
NOTE: For this protocol, peripheral whole blood samples are collected by venipuncture in lithium-heparin collection tubes from healthy adult volunteers (with informed consent - BM18/6/12 Biomedical Research Ethics Committee of The University of Western Cape Ethics Committee). Before beginning, ensure that the blood and the density medium of 1.077 g/mL are acclimated to room temperature.
<ol>
	<li>In a prepared biosafety cabinet, dilute the peripheral whole blood in a 1:1 volume with PBS and gently layer the diluted blood on an equal volume to two-volumes of medium with a density of 1.077 g/mL. It is important to gradually layer the blood on the density medium by holding the tube slanted at 45&#176;, ensuring that the blood and density medium do not mix.</li>
	<li>Carefully transfer the suspension to a centrifuge and spin at 900 x g for 20 minutes. Thereafter, pipette the &#39;cloudy&#39; layer only to a new sterile conical tube and fill the tube with PBS and centrifuge for 10 minutes at 1000 x g. Thereafter, aspirate the supernatant with a pipette, being cautious to not disturb the pellet, resuspend the PBMC pellet carefully in 1 mL PBS and fill the tube with PBS.</li>
	<li>Repeat the above washing step two more times to get to a total of three washes.</li>
	<li>Count the number of PBMCs using a hemocytometer, aware that each mL of peripheral blood from an adult donor will result in a rough average of 1 - 1.5 x 106 PBMCs49. After counting, dilute the PBMC pellet to a concentration of 8 x 105 lymphocytes in 1 mL of cRPMI. Then add approximately 2 x 106 PBMCs or 2.5 mL from the isolated PBMC solution into a sterile, conical tube for irradiations.</li>
</ol>
3. Sample irradiations
CAUTION: Handle the radiation unit according to Radiation Protection guidelines and wear a physical dosimeter at all times. Ensure that the irradiation system used is calibrated and set up such that the correct expected doses are achieved.
<ol>
	<li>Irradiate PBMCs at room temperature using a 60Co source. Calibrate (TRS-398 protocol) with a Farmer 117 chamber for which a chamber calibration factor obtained from the National Metrology Institute of South Africa (NMISA)50.
	<ol>
		<li>For this assay, place the conical tubes between a 5 mm acrylic sheet (used for beam entry build-up) and 50 mm backscatter material with the current 60Co source dose rate of 0.57 Gy/min for a 300 mm &#215; 300 mm homogenous field size at 750 mm Source to Surface Distance.</li>
	</ol>
	</li>
	<li>Irradiate the PBMCs with graded doses of 0.125, 0.500, 1.000, 1.500 and 2.000 Gy, and maintain the sham-irradiated control samples, in the control room, receiving only ambient radiation exposure.</li>
	<li>Incubate the irradiated samples for 1 hour at 37 &#176;C in a humidified 5% CO2 incubator in order to reach the maximum number of &#947;-H2AX foci. 
	&#8203;NOTE: Incubation time can be modified according to experimental design.</li>
</ol>
4. Slide preparation
NOTE: See Figure 1 for slide setup.
<ol>
	<li>Prepare 3 slides (technical repeats) using slides coated with a positively charged surface to improve adhesion per irradiation condition (or per conical tube). Place the slide into the clip holder, add the filter card on top of it, and finally the funnel. Secure the slide clips and place into the cytocentrifuge.</li>
	<li>Add 250 &#181;L of the cell suspension to the funnel (200,000 cells/slide) and spin for 30 x g for 5 minutes using a cytocentrifuge.</li>
	<li>After cytocentrifugation, remove the slide from the clip holder and using a hydrophobic pen, draw a circle around the spot with the cells in order to retain the immunofluorescent stain on the bound cells during the staining process.</li>
</ol>
5. Fixation and immunofluorescence &#947;-H2AX staining
NOTE: All solutions are carefully added directly onto the cell area which is marked by a hydrophobic circle. Staining solutions are dispensed slightly above the slide without allowing the pipette tip to disrupt the cells. Solutions are NOT added to the whole slide.
<ol>
	<li>Fix the slides in freshly prepared 3% PFA for 20 minutes. 
	NOTE: The slides can be stored in PBS containing 0.5% PFA at 4 &#176;C for a maximum of 2 days before immunostaining is performed. Thereafter, wash slides in a Coplin jar with PBS for 5 minutes, then cover the cells with 100 &#181;L of the cold Triton-X solution for 10 minutes to allow permeabilization.</li>
	<li>Tip the Triton-X solution off on tissue paper and wash the slides three times in 1% BSA solution for 10 minutes. This is done to prevent unwanted background by blocking nonspecific antibody binding.</li>
	<li>Incubate slides at room temperature with 100 &#181;L of the 1:500-diluted primary anti-&#947;-H2AX antibody for 1 hour in a humidifying chamber, easily accomplished by adding wet tissue paper at the base of a rectangular slide storage box.
	<ol>
		<li>Tip the antibody solution off on tissue paper and perform three washes with 1% BSA solution for 10 minutes in a Coplin jar to remove unbound primary antibody and to prevent non-specific binding of the secondary antibody.</li>
	</ol>
	</li>
	<li>Incubate slides with 100 &#181;L of the 1:1000-diluted secondary DAM-TRITC antibody solution for 1 hour in the humidifying chamber. Tip the antibody solution off on tissue paper and wash the slides in PBS for 10 minutes three times.</li>
	<li>Finally dry the slides outside the hydrophobic circle with soft, dust-free tissue paper and add&#160;1-2 drops of DAPI resolved in an aqueous mounting medium and gently cover the slide with a 24 x 50 mm cover slip, ensuring that there are no trapped air bubbles and place in the fridge at 4 &#176;C overnight. Slides can be stored at 4 &#176;C for a maximum of 2 weeks before scanning.</li>
</ol>
6. Automated Scanning and Scoring of slides
NOTE: The scanning system needs to have a fluorescent microscope, a high-resolution CCD (charged coupled device) camera connected to a frame grabber for real-time digitization of video images, a scanning stage, trackball for manual movements, 3D mouse, fast PC and monitor, and hard drive for archiving (Figure 2).
<ol>
	<li>Classifier setup 
	NOTE: The classifier is a set of parameters defining how the system detects cells. It results from the training based on pre-classified image fields. A classifier is specific for a microscope magnification, cell type, and laboratory. Classifiers may therefore require modifications in the following parameters to adapt them to the current conditions. It is recommended to test the settings with a reference slide before evaluation.
	<ol>
		<li>Image acquisition: Use a 40x dry objective for imaging. To achieve comparable image quality in all cells, set the camera integration time to automatic mode. Set the maximum integration time to at least 1 second for both color channels (camera gain 4.0). The signal channel is acquired with 10 focus planes and 14/40 &#181;m between the planes.</li>
		<li>Cell selection: PBMCs within a range of 20.00 &#181;m2 and 76.00 &#181;m2 are detected. Set the maximum concavity depth to 0.05 and the maximum aspect ratio to 1.4. Set the relative gray level threshold to 20%.</li>
		<li>Cell processing: Process the DAPI counterstain channel with a histogram maximum algorithm to reduce the background. The signal channel is processed with a TopHat filter (strength 7, smoothing factor 0) to reduce the background and to enhance the signals.</li>
		<li>Signal counting: Count foci using the Object Count feature of the device. To avoid false-positive evaluation of remaining background signals, only those signals which show at least 30% of the intensity when compared to the brightest object in the cell are counted.</li>
	</ol>
	</li>
	<li>Insert/Place slides onto the automated scanning platform or slide stage, after gently cleaned with 70% ethanol, to remove any dust.</li>
	<li>Slide Setup - Open Slide Setup Dialogue Menu (Figure 3)
	<ol>
		<li>Select the correct Data Path, this specifies where the slide files resulting from the search are stored.</li>
		<li>Give the slide a name, each slide must be given a unique name. If a series of slides are entered in the form of an enumerated list, the &#39;?&#39; can be used as a wildcard for the slide number.</li>
		<li>Select the appropriate classifier, as described in section 1.1 - 1.4 (Figure 4).</li>
		<li>Select the Search Window and select Predefined if a search window definition matching the cytocentrifuge cell circle exists, select the respective definition in the Size column, or select Manual if there is no predefined search window definition available. In this case, the Size column does not have to be set.</li>
		<li>Select Maximum Cell Count and add the maximum number of cells required to be scanned. The search is terminated even if the selected search window has not yet been scanned completely as soon as the Max Cell Count is reached. For biodosimetry applications, automated scoring of 1000 cells per slide is sufficient, considering that 3 slides per blood sample are prepared. Click OK to confirm the settings.</li>
	</ol>
	</li>
	<li>Slide Scanning Setup
	<ol>
		<li>Click the Search button on the side bar. If the setting Manual Search Window was selected in slide setup, a dialogue will open allowing for determination of the scan area (Figure 5). By using the 10x objective, select the rectangular search area on the slide interactively by fixing two corners of the search field by left click of the mouse. This can be confirmed with the OK button. If the search window is referring to a predefined search window, this step will be omitted.</li>
		<li>The user is prompted to adjust a focus start position. A reference object will then be selected automatically, the software will prompt the user to focus and center a reference nucleus (using the 40x objective) for each slide and to confirm settings with OK. The system will automatically move to all slides sequentially. If necessary, adjust Live Image Setup - Integration Time for this step (Figure 5).</li>
		<li>Check all microscope settings and confirm the system prompt with OK. The system will start the automated focusing and scanning procedure. Once scanning is completed the data is saved to the computer for analyses. The scanning system automatically switches off after the scan is complete. 
		NOTE: Make sure that the lever of the microscope tube is in a position that diverts all light to the camera.</li>
		<li>Search End, the search is terminated if the whole search has been scanned, if the maximum cell count has been reached or if the search has been canceled.</li>
	</ol>
	</li>
	<li>Slide Analysis 
	NOTE: The completed scan is represented in Figure 6.
	<ol>
		<li>When the scan is completed, click the Gallery button on the side bar. Review the gallery, which consists of small images of detected cells. In this window (Figure 7), specify if the cells stored in the gallery by a criterion to be chosen from a drop-down menu. Cells are generally displayed in the order in which they have been detected.</li>
		<li>Click on Quality Histogram/Feature Value Diagram (Figure 8) to give the distribution of the quality measure for the detected cells, as well as the means, and the standard deviation of foci scored.</li>
	</ol>
	</li>
</ol>

C. Schunck is an employee of MetaSystems Hard &amp; Software GmbH, Altlussheim, Germany.

This protocol describes a stepwise procedure for&#160;the&#160;automated fluorescent microscopy-based scoring of the&#160;&#947;-H2AX&#160;foci assay. It illustrates the utility of the foci assay as a time efficient method for analyzing the number of radiation-induced DNA DSB&#160;in peripheral blood lymphocytes to perform a biological dose assessment in a radiation accident scenario&#160;where individuals might be exposed to unknown levels of IR.
In this specific protocol, PBMCs were irradiated in vitro to mimic an in vivo radiation exposure. Once the irradiation and the incubation time of one hour is completed, slides are made using a cytocentrifuge to create a concentration spot of cells on the slide. The use of a cytocentrifuge is vital to achieve standardized conditions for automated scoring. When completed, a hydrophobic pen is used to make a circle around the cells, to reduce wastage of reagents by allowing the user to localize the staining reagents. This type of pen can be used in various immunostaining techniques such as on paraffin sections, frozen sections and cytology preparations. Furthermore, it is important to select a hydrophobic pen which is compatible with enzyme and fluorescent-based detection systems. Following slide preparation, the fixation and immunofluorescence &#947;-H2AX staining occurred. In this protocol, the cells are fixed using 3% PFA in a PBS solution for 20 minutes. For immunostaining to succeed, it is essential that the morphology of the cells is retained and that the antigenic sites are accessible to the detection reagents being used. PFA is a relatively gentle agent for fixation and stabilizes cells while preserving protein structures51. Optimization experiments with higher PFA concentrations and longer fixation times resulted in a negative impact on the slide quality, but further storage (overnight) in 0.5% PFA up to 24 hours yielded good results.
The primary, 2F3 monoclonal antibody used in this protocol reacts to histone variant H2AX when phosphorylated at Serine 139 after DNA DSB induction. The antibody is able to bind to the phosphorylated residue with no cross reactivity with other phosphorylated histones52. Since this is a primary mouse monoclonal antibody, a secondary antibody was selected against the host species of the primary antibody while raised in an alternative host, namely donkey-anti-mouse (DAM)-TRITC. While immunofluorescent staining is based on specific antibody-epitope binding, several intermolecular forces can also result in non-specific background staining. In order to reduce non-specific binding, it is important to use a blocking reagent in immunofluorescent staining protocols53; we used a BSA solution. Furthermore, sufficient time should be allocated to this blocking step by leaving the slides in the solution for at least 20 minutes prior to primary and secondary antibody staining. In addition, the BSA solution should also be used as diluent for the primary and secondary antibodies. Depending on the anti-&#947;-H2AX and secondary antibody that is used for the staining, one should consider testing different antibody dilutions in order to determine the optimal concentration. For more precise scoring, a double staining can be conducted, by adding additional DNA DSB repair protein antibodies.
A major disadvantage of this type of analysis is the need to acquire blood samples as soon as possible after exposure, as the maximum number of foci is known to decrease back to normal levels within 48 hours post irradiation. Therefore, when the time of radiation accident and subsequent blood sampling is known, it could be useful to work with different calibration curves that have been established at different time points after in vitro irradiation (e.g., 4, 8, 12 and 24 hours). However, as already mentioned in the introduction section of the manuscript, the strength of the &#947;-H2AX foci assay lies in initial, fast triage purposes and it should be used to prioritize more time-consuming cytogenetic biological dosimetry. A combined scenario where multiple biodosimetry biomarkers are used in parallel, will generate the most reliable dose estimation and various biodosimetry laboratories worldwide have joined forces to set up nationwide networks that can be activated and used to allow multiple, parallel biodosimetry assessments by laboratories with different expertise37,54,55. In addition, developments are ongoing for superfast analysis such as a mobile laboratory on or near the accident site56. New, promising biodosimetry methods are constantly being developed, which will hopefully result in even faster and more reliable throughput in the future57.
For the automated image analysis system, slides are inserted or placed on the automated scanning platform or slide stage. Thereafter, name and save the slide details in the appropriate folder on the attached computer. For this experiment, automated nuclei and foci detection is based on the respective classifier settings. On creating a classifier, ensure that the selected classifier settings match the current cell type, preparation conditions, and immunofluorescent staining of the sample. Appropriate fluorescent channels matching the excitation spectrum of the primary and secondary antibodies are set in the classifier. The classifier allows for setting additional scoring parameters if required (e.g., nucleus size, fluorescent intensity, as described in Section 6.1). If two or more DNA repair proteins (e.g., &#947;-H2AX and 53BP1) are combined in an experiment, the system is also capable of detecting co-localizations of signals. First, the system acquires DAPI images, applies image processing, and identifies nuclei using morphological criteria set in the classifier. The TRITC signals are acquired using 10 z-stacks with a step size of 0.35 mm between the focal planes47. The classifier used the Direct Foci Count, where the number of distinct TRITC signals within the nucleus are scored. Here, it is important to take into consideration that with increasing radiation dose, foci signals tend to merge into larger objects, resulting in an underestimation of the actual foci number if objects are counted directly. It was not required for the analysis described here, but an additional step with Corrected Foci Count can be implemented to solve this problem. The latter allows the system to obtain the sizes of the detected signals and weighs them accordingly. Using both counting methods can provide a more realistic estimation of the actual number of foci at higher doses.
To begin automated scanning, the scan area is determined by using the 10x objective of the microscope to make a rectangular search area by fixing two corners of the search field by left click of the mouse (Figure 5), followed by focusing the start position. The reference object is selected automatically, and the software prompts the user to focus and center a reference nucleus (using the 40x objective) for each slide. After the search has begun, the system will move to the center of the search window of the first selected slide and will request to center and focus the reference object. This object will later be used as a position reference to correct any shift in the cell&#39;s positions. The second purpose of the reference field is the automatic light adjustment, in transmitted light mode the light is adjusted until the optimum light level is reached. In fluorescence mode the light level is fixed, but the integration time of the CCD camera can be increased until the required signal is measured. To enable a correct light adjustment, the reference should contain objects with typical staining. It is important to not use a field which has artifacts with very high staining intensity. Following the light adjustment, the system starts the grid autofocus at the grid position closest to the reference field. It continues to focus fields on a regular grid, moving in a meander towards the front and the back of the search window. Scanning begins when the grid autofocus is completed. The stage is moved in a meander pattern field after field to capture data. When a cell is detected, its position and gallery image are stored and displayed on the screen and the cell count is updated. If a microscope, stage or feeder error occurs, the search is automatically canceled. The only step where there is a manual intervention from the operator, is&#160;during the slide scanning set-up. This is also the point where a quick quality control check takes place (air bubbles, low cell numbers, fading of the fluorescent signal staining artefacts) and where it can be decided to abort the scanning of a slide of inferior quality. A search is terminated if the whole slide has been scanned, if the maximum cell count has been reached, or if the search has been canceled. Once the scan is completed, the data is presented as seen in Figure 6. To view scanned cells, the Gallery window is opened and each cell can be viewed (Figure 7). This is another point where the operator can perform a quality control by checking the focus of the gallery images and the total number of cells that have been scored. If too many cells are out of focus or too little cells were detected by the system to make a realistic dose estimation (e.g., 100 cells instead of the intended 1000 cells), then the decision should be made to exclude the slide and the automatic score from the final evaluation. All data are summarized in histograms (Figure 8), together with information on the distribution, the means, and the standard deviation of foci scored for each cell. The histograms can also be used to select and display sub-populations of nuclei based on the automated findings for review. Statistical analyses on the results are performed after the distribution, mean, and the standard deviation of the number foci per cell have been recorded manually. The graph can be used as a calibration curve for making a dose estimation of a biodosimetry sample. This can be done using the equation of the trend line to make an approximate estimation of the dose received. Moreover, Figure 9 illustrates that the automated scanning is sensitive enough to detect foci induced at low doses. Furthermore, the results show a clear linear increase of the number of foci per cell with dose. It should be noted that the results are only representative for the used classifier, results will differ for different classifier parameters. Therefore, in case of a biodosimetry analysis, it is important that the same classifier and slide preparation are used for the biodosimetry samples as the ones that have been used to establish the calibration curve that is used to perform the dose estimation. While it was out of the scope of this study, it is important to note that the &#947;-H2AX foci assay can also be used to determine partial body irradiations. Most accidental radiation exposures are inhomogeneous or partial body exposures, where only a localized region of the body received a high dose exposure. Several studies illustrated that it is possible to use the &#947;-H2AX foci assay to estimate the fraction of the body that has been irradiated and the dose to the irradiated fraction42. When a whole-body irradiation takes place, there will be random induction of DNA DSB&#160;in all cells and one can expect to find a Poisson distribution. Similar to cytogentic methods where the induction of chromosomal aberrations tends to be over dispersed in peripheral blood lymphocytes where there is a high abundance of cells with multiple aberrations and cells with normal metaphases, dispersion analysis of &#947;-H2AX foci using a contaminated Poisson method suggested over dispersed foci distributions58. The latter was also confirmed in in vivo experiments with minipigs and a rhesus macaque59.

Since its discovery, ionizing radiation (IR) has become an indispensable tool in current medical and industrial practices, as well as in agricultural and military applications. However, the wide use of IR also increases the risk of radiation overexposure for both professional radiation workers as well as members of the public. IR is a well-known physical carcinogen that can induce DNA damage in a direct or indirect manner, leading to significant health risks 1,2. Therefore, it is important to perform a dose assessment, since the degree of exposure forms an important initial step in the management of the radiation accident 1.
In the event of large scale nuclear or radiological emergencies, the number of dose assessments that need to be performed can range from a few to thousands of people depending on the size of the accident3. In these scenarios, physical dosimetry may also be ambiguous (e.g., if the dosimeter is not worn properly) or unavailable, when members of the public are involved. While clinical symptoms could be used for triage, they are not necessarily radiation specific and can result in a false diagnosis. Therefore, it is advisable to use biological dosimetry alongside physical dosimetry and clinical assessments. This method allows the analysis of radiation-induced changes at cellular level and enables the unequivocal identification of exposed individuals who require medical treatment4. The physician can then use this biological dose assessment to complement physical dose reconstructions and other clinical diagnoses, in order to prescribe the appropriate medical care5. The need for biological dosimetry will be especially high for triage and medical management of casualties when the exposure scenario is not well known and when the victims are members of the public. The main purpose of triage is to effectively distinguish &#34;worried well&#39; persons, who could have prodromal symptoms but who did not receive a high dose, from exposed persons needing immediate medical help and specialized care. The threshold level of radiation dose that can cause radiation sickness is approximately 0.75 - 1.00 Gy. Then, those individuals who receive &#62; 2 Gy of exposure are at higher risk for acute radiation syndrome and should receive prompt medical treatment6,7. Timely and accurate biological dose estimates for victims caught in the crossfire of such disasters are critical. Additionally, it can also comfort and reassure minimally exposed victims8.
Radiation protection authorities use various biodosimetry markers, which are mainly based on the detection of cytogenetic damage, such as dicentric chromosomes or micronuclei, in cultured human lymphocytes9. The detection of cytogenetic damage can also be performed by the fluorescent in situ hybridization (FISH) translocation assay10. However, the major drawback of the conventional cytogenetic methods is the long turnaround time to obtain the results in an emergency situation8.
One of the earliest steps in the DNA DSB recognition process is the phosphorylation of the histone variant H2AX, leading to the formation of &#947;-H2AX, and the subsequent recruitment of repair factors. Over the past decade, the detection of radiation-induced &#947;-H2AX foci in peripheral blood lymphocytes using immunofluorescence microscopy has received growing attention as a reliable biological dosimetry tool11,12,13,14,15. Depending on the radiation quality and cell type, the maximum yield of &#947;-H2AX foci is detected within 0.5 - 1 hour after irradiation16,17. It is anticipated that there is a close correlation between the number of DNA DSB and &#947;-H2AX foci, as well as between the disappearance of foci and DSB repair. Laser scissor experiments with a pulsed laser microbeam have demonstrated that &#947;-H2AX foci localize to the sites of DNA DSB18. While it remains a topic of active debate, one of the earliest studies with 125I suggested a one-to-one correlation between the calculated number of disintegrations per cell (representable for the number of radiation-induced DNA DSB) and the number of &#947;-H2AX foci that was scored19,20.
Over the past decade, the European Union funded the MULTIBIODOSE (Multi-disciplinary biodosimetric tools to manage high scale radiological casualties) and RENEB (Realizing the European Network of Biodosimetry) projects to create a sustainable network in biological and retrospective dosimetry21,22. This project included various laboratories throughout Europe to evaluate the emergency response capabilities in the event of a radiological emergency14,21,22. The &#947;-H2AX foci assay has a number of considerable advantages, such as a rapid processing time, the potential for batch processing allowing for high throughput, as well as its high sensitivity if used within a few hours after exposure13,23,24. The high sensitivity of the assay in the low dose range resulted in a number of studies where the &#947;-H2AX foci assay was used as an indicator of the effect of medical radiation exposure, both in radiotherapy as well as in diagnostic imaging applications25,26,27,28,29,30. These characteristics make the &#947;-H2AX foci assay a highly competitive alternative to other methods for early triage in large nuclear accidents to separate the critically exposed from low risk individuals. Several optimization experiments have illustrated that it is feasible to perform the &#947;-H2AX foci assay with small volumes of blood, such as the study of Moquet et al. who reported that it is feasible to perform the &#947;-H2AX foci assay with only a drop of blood (finger prick)31. A similar approach was used in the development of the fully automated high-throughput RABIT (Rapid Automated Biodosimetry Tool) system which was optimized to measure &#947;-H2AX yields from fingerstick-derived samples of blood32,33. Overall, the results of the MULTIBIODOSE and RENEB inter-comparison studies suggest that the &#947;-H2AX foci assay could be a very useful triage tool following a recent (up to 24 hours) acute radiation exposure12,13,14. In a low-dose range inter-comparison study, a dose as low as 10 mGy could be distinguished from the sham-irradiated control samples, highlighting the sensitivity of the assay in the low dose range34. It is important to note that the high sensitivity of the assay is particularly true for lymphocytes, which makes them one of the most suitable cell types for the evaluation of low-dose exposures. Lymphocytes are mainly non-cycling cells and represent a synchronous population. The latter avoids expression of &#947;-H2AX is associated with DNA replication, which significantly reduces the assay&#39;s sensitivity to detect radiation-induced DSB during G2 and S-phase of the cell cycle35. In addition to its sensitivity to low doses for lymphocytes, the turnaround time of the &#947;-H2AX foci assay is significantly faster than cytogenetic techniques such as the dicentric and micronucleus assay, since it does not require the stimulation of lymphocytes. Therefore, results can be obtained within a few hours compared to a couple of days for cytogenetic techniques. A major drawback of the assay is the fast disappearance of the &#947;-H2AX foci signal, which will normally be reduced to baseline levels within days after the radiation exposure depending on the DNA repair kinetics36. Therefore, the most suitable application of the assay in a biodosimetry context is for initial triage purposes and to prioritize more time-consuming cytogenetic biological dosimetry follow-up for certain victims. However, for precise retrospective biodosimetry and long-term effects, one has to rely on cytogenetic techniques such as three-color FISH analyses for the detection of stable chromosomal aberrations in case the exposure took place several years ago10.
As part of several biodosimetry initiatives, multiple assays&#160;have been evaluated for triage purposes next to the &#947;-H2AX foci assay to triage people in large-scale radiological emergencies; such as the dicentrics assay, the cytokinesis-block micronucleus assay, electron paramagnetic resonance (EPR), serum protein assay (SPA), skin speckle assay (SSA), optically stimulated luminescence (OSL), as well as gene expression analysis 37,38. The &#947;-H2AX foci assay can be used quantitatively for the evaluation of DNA DSB formation and repair39. However, the assay is time-dependent as the level of &#947;-H2AX foci varies with time after irradiation due to the DNA DSB repair kinetics40. A comparative study illustrated that microscopic scoring with z-stage capacity offers the most accurate results after 1 Gy of irradiation, while flow cytometry only gives reliable results at higher doses of IR41. There are many reports of the development of image analysis solutions for use in automated scoring42,43,44,45,46. In this protocol, an automated, high-throughput fluorescent microscopy platform is used to analyze &#947;-H2AX foci in peripheral blood lymphocytes. The use of an automatic scoring system avoids both inter-laboratory and inter-observer scoring bias, while it still allows enough sensitivity to detect doses below 1 Gy47.&#160;The primary advantage of this system compared to free open-source software for foci scoring is the fact that the complete process from the scanning of slides to capturing and scoring is automated. The concept of user-definable and storable classifiers guarantees reproducibility which adds an unbiased degree of quality to the results. Therefore, this work illustrates how &#947;-H2AX foci results can be obtained using an automated microscopic scanning and scoring method which can be used when blood samples from potentially over-exposed individuals are received by radiobiology laboratories for biological dosimetry purposes.&#160;

<table><tbody><tr><td>Bovine serum albumin - BSA</td><td>Merck</td><td>A3059</td><td></td></tr><tr><td>Coplin Jar</td><td>Sigma</td><td>S6016</td><td></td></tr><tr><td>Cover Slips (Size 24 x 50 mm)</td><td>Lasec</td><td>Glass2C29M2450Rec</td><td></td></tr><tr><td>Cryogenic Vials (1.2 mL)</td><td>WhiteSci</td><td>607101</td><td></td></tr><tr><td>Cytospin</td><td>Healthcare Technologies</td><td>JC370-12-L</td><td></td></tr><tr><td>Cytospin Clips</td><td>Healthcare Technologies</td><td>JC302</td><td></td></tr><tr><td>Cytospin filter cards</td><td>Healthcare Technologies</td><td>JC307</td><td></td></tr><tr><td>Cytospin Funnels</td><td>Healthcare Technologies</td><td>JC372</td><td></td></tr><tr><td>DAM-TRITC</td><td>Invitrogen</td><td>A16022</td><td></td></tr><tr><td>DAPI-Fluroshield</td><td>Sigma/Merck</td><td>F6057</td><td></td></tr><tr><td>Ethanol</td><td>Kimix</td><td>ETD901</td><td></td></tr><tr><td>Filter tips</td><td>WhiteSci</td><td>&amp;nbsp;200ul (312012) and 1000ul (313012)</td><td></td></tr><tr><td>Hydrochloric acid- HCl</td><td>Merck</td><td></td><td></td></tr><tr><td>Histopaque</td><td>Sigma/Merck</td><td>10771</td><td></td></tr><tr><td>lithium&amp;ndash;heparin collection tubes</td><td>The Scientific Group</td><td>367526</td><td></td></tr><tr><td>MetaSystems - Metafer</td><td>Metasystems</td><td>Azio Imager Z2: 195-041848</td><td></td></tr><tr><td>NaOH</td><td>Merck</td><td>221465</td><td></td></tr><tr><td>NovoPen</td><td>Leica Biosystems</td><td>NCL-PEN</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma/Merck</td><td>158127</td><td></td></tr><tr><td>Phosphate-buffered saline Tablets</td><td>Separations</td><td>SH30028,02</td><td></td></tr><tr><td>Pipettes</td><td>WhiteSci</td><td>P11037 and P1033</td><td>X-tra Clipped Corner Slides are clipped at 45&amp;deg; angles to help reduce glass breakage.</td></tr><tr><td>Purified anti-H2A.X Phospho (Ser139) Antibody</td><td>Biolegend</td><td>613402 / 100 &amp;mu;g</td><td></td></tr><tr><td>RPMI medium</td><td>WhiteSci</td><td>BE12-702F</td><td></td></tr><tr><td>Triton X-100 (Octoxinol 9)</td><td>Sigma/Merck</td><td>T-9284</td><td></td></tr><tr><td>Tubes (15ml)</td><td>WhiteSci</td><td>601002</td><td></td></tr><tr><td>X-Tra adhesive slides &amp;ndash; corner clipped</td><td>SMM Technologies</td><td>3800200E</td><td></td></tr></tbody></table>

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.

For this protocol, human peripheral blood mononuclear cells (PMBCs) were isolated from whole blood of four healthy adult volunteers and were exposed to radiation doses of 0.125, 0.250, 0.500, 1.000 and 2.000 Gy. Thereafter, the samples were incubated for 1 hour at 37 &#176;C in a humidified 5% CO2 incubator. Following fixation and immunofluorescence staining, the slides are automatically scanned and scored. Figures 6, 7, and 8 give a representation of what the software outputs to the user. An analysis window appears once the scan is completed, giving an overview of the foci scored. The gallery gives all foci scored with an interactive menu that can delete outliers and finally a histogram with a detailed data summary is given.
The &#947;-H2AX foci yield after exposure to &#947;-rays are presented in Figure 9. There was a gradual increase in the number of &#947;-H2AX foci with increasing dose. This graph does not only illustrate the dose dependence and sensitivity of the assay, but the fit can also be used to perform a dose estimation when a sample is received from an individual who has been exposed to an unknown dose. It is important to note that one will not have sham-irradiated control or background foci levels for this individual. Therefore, Figure 9 includes the 0 Gy value of the sham-irradiated control samples, which was 0.57&#160;&#177;&#160;0.23&#160;Gy for the four adult donors in this study.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62623/62623fig01.jpg" /> 
Figure 1: Preparation of slides for cytocentrifugation. Place the slide into the clip holder, add the filter card on top of it, and finally the funnel. Secure the slide clips and place into the cytocentrifuge. After cytocentrifugation, remove the slide from the clip holder and draw a circle around the spot with the cells using a hydrophobic pen. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62623/62623fig01large.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/62623/62623fig02.jpg" /> 
Figure 2: The automated scanning platform that was used in this protocol, containing an automated scanner attached to a fluorescent microscope. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62623/62623fig02large.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/62623/62623fig03.jpg" /> 
Figure 3: Menu for slide setup. Open the dialogue by clicking the SETUP button in the sidebar. Select or enter the target directory for the result files <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62623/62623fig03large.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/62623/62623fig04.jpg" /> 
Figure 4: Classifier Setup Menu. Ensure that the selected classifier settings match the current cell type, preparation conditions, and staining patterns of the sample. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62623/62623fig04large.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/62623/62623fig05.jpg" /> 
Figure 5: Setup menu for Manual Search Window. Window was selected in slide setup; a dialogue will open allowing for determination of the scan area. By using the 10x objective, the rectangular search area on the slide is selected interactively by fixing two corners of the search field by left click of the mouse. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62623/62623fig05large.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/62623/62623fig06.jpg" /> 
Figure 6: Analysis Window, once the scan is completed, the analysis window shows the results of the scan. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62623/62623fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/62623/62623fig07.jpg" /> 
Figure 7: Gallery View for the selected slide. The Gallery tab is found on the side bar. The image gallery consists of small images of the cells detected, displayed as a combined DAPI-TRITC image. In the lower left and right corner of each image the direct foci count and the corrected foci counts are displayed. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62623/62623fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/62623/62623fig08.jpg" /> 
Figure 8: Histogram images for the selected slide. This window is opened by right clicking the histogram. By clicking on the histogram, the histogram tab sheet provides a data summary which lists the mean, standard deviation, coefficient of variation, minimum, maximum and median values of the analyzed cells. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62623/62623fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/62623/62623fig09.jpg" /> 
Figure 9: The number of &#947;-H2AX foci as a function of dose for lymphocytes exposed to 60Co &#947;-rays. The error bars are the standard deviations representing the interindividual variation among the donors. Data represents the mean number of &#947;-H2AX foci &#177; standard deviation of four healthy volunteers. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62623/62623fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about An Automated Microscopic Scoring Method for the Î³-H2AX Foci Assay in Human Peripheral Blood Lymphocytes at JoVE.com

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

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 ...

an-automated-microscopic-scoring-method-for-h2ax-foci-assay-human

Nanyang Technological University

Research

JoVE Journal

Cancer Research

4.7K Views.  iThemba LABS. Mijn naam is Dr. Shankari Nair en ik ben een postdoctoraal fellow bij de afdeling Stralingsbiofysica van de afdeling Nucleaire Geneeskunde van NRF, iThemba LABS. In deze video demonstreren we het gebruik van een geautomatiseerde Gamma H2AX foci assay op menselijke perifere bloedlymfocyten voor het gebruik ervan in batch symmetrie toepassingen. De stralingsrisico's voor het personeel worden streng gecontroleerd.Ioniserende straling beïnvloedt direct de structuur van DNA door DNA-breuke...

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

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 ...

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

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair complexes. This process is regulated by a myriad of proteins that promote the association and disassociation of proteins to these lesions. Thanks in large part to the ability to perform functional screens of a vast library of proteins, there is a greater appreciation of the genes necessary for the double-strand DNA break repair. Often knockout or chemical inhibitor screens identify proteins involved in repair processes by using increased toxicity as a marker for a protein that is required for DSB repair. Although useful for identifying novel cellular proteins involved in maintaining genome fidelity, functional analysis requires the determination of whether the protein of interest promotes localization, formation, or resolution of repair complexes. 
The accumulation of repair proteins can be readily detected as distinct nuclear foci by immunofluorescence microscopy. Thus, association and disassociation of these proteins at sites of DNA damage can be accessed by observing these nuclear foci at representative intervals after the induction of double-strand DNA breaks. This approach can also identify mis-localized repair factor proteins, if repair defects do not simultaneously occur with incomplete delays in repair. In this scenario, long-lasting double-strand DNA breaks can be engineered by expressing a rare cutting endonuclease (e.g., I-SceI) in cells where the recognition site for the said enzyme has been integrated into the cellular genome. The resulting lesion is particularly hard to resolve as faithful repair will reintroduce the enzyme's recognition site, prompting another round of cleavage. As a result, differences in the kinetics of repair are eliminated. If repair complexes are not formed, localization has been impeded. This protocol describes the methodology necessary to identify changes in repair kinetics as well as repair protein localization.

Repair of double-strand DNA breaks is a dynamic process, requiring not only formation of repair complexes at the breaks, but also their resolution after the lesion is addressed. Here, we use immunofluorescence microscopy for transient and long-lasting double-stranded breaks as a tool to dissect this genome maintenance mechanism.

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair ...

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

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

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

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

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 ...

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 ...

Extraction and Identification of Micronuclei from Human Peripheral Blood Lymphocytes

A micronucleus (MN) is an abnormal nuclear structure that forms in cells, particularly bone marrow or blood cells, when exposed to external damage, such as radiation, due to unresolved DNA damage or mitotic errors. Once formed, MNs can actively contribute to various carcinogenic processes, including inflammatory signaling and chromosomal genetic rearrangements. MNs contain nuclear DNA, histones, nucleoprotein fragments, and other active proteins, which are closely associated with their functions. Studying the formation and components of MNs is crucial for understanding their role in driving carcinogenic processes. The extraction and purification of MNs are essential to achieving these research objectives. However, the instability of the MN nuclear membrane and its susceptibility to rupture make these processes technically challenging. Currently, only a few studies have reported the use of density gradient centrifugation for MN separation. This study summarizes and simplifies the processes of MN separation and purification. Human peripheral blood lymphocytes exposed to radiation were isolated, and MNs were separated and purified using sucrose buffers of different concentrations in a two-step process. The integrity and purity of the MNs were verified, providing a clear and practical demonstration of the experimental procedure for researchers investigating the causes and functions of MNs.

This protocol outlines a method for extracting and purifying micronuclei from human lymphocytes using sucrose density gradient centrifugation. It provides an experimental basis for investigating the composition and function of micronuclei.

A micronucleus (MN) is an abnormal nuclear structure that forms in cells, particularly bone marrow or blood cells, when exposed to external damage, such ...

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

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