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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>&#160;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&#8211;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&#176; angles to help reduce glass breakage.</td>
		</tr>
		<tr>
			<td>Purified anti-H2A.X Phospho (Ser139) Antibody</td>
			<td>Biolegend</td>
			<td>613402 / 100 &#956;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 &#8211; 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/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/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/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/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/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/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/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/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/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 ÃŽÂ³-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 ...

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JoVE Journal

Cancer Research

4.7K Views.  iThemba LABS. This protocol presents the slide preparation and automated scoring of the γ-H2AX foci assay on peripheral blood lymphocytes. To illustrate the method and sensitivity of the assay, isolated lymphocytes were irradiated in vitro. This automated method of DNA DSB detection is useful for fast and high-throughput biological dosimetry applications.

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

Intra-iliac Artery Injection for Efficient and Selective Modeling of Microscopic Bone Metastasis

Intra-iliac artery (IIA) injection is an efficient approach to introduce metastatic lesions of various cancer cells in animals. Compared to the widely used intra-cardiac and intra-tibial injections, IIA injection brings several advantages. First, it can deliver a large quantity of cancer cells specifically to hind limb bones, thereby providing spatiotemporally synchronized early-stage colonization events and allowing robust quantification and swift detection of disseminated tumor cells. Second, it injects cancer cells into the circulation without damaging the local tissues, thereby avoiding inflammatory and wound-healing processes that confound the bone colonization process. Third, IIA injection causes very little metastatic growth in non-bone organs, thereby preventing animals from succumbing to other vital metastases, and allowing continuous monitoring of indolent bone lesions. These advantages are especially useful for the inspection of progression from single cancer cells to multi-cell micrometastases, which has largely been elusive in the past. When combined with cutting-edge approaches of biological imaging and bone histology, IIA injection can be applied to various research purposes related to bone metastases.

This manuscript provides the detailed procedure of intra-iliac artery (IIA) injection, a technique to deliver cancer cells specifically to hind limb tissues including bones to establish experimental bone metastases. Although initially established with breast tumor models, this protocol can be easily extended to other cancer types.

Intra-iliac artery (IIA) injection is an efficient approach to introduce metastatic lesions of various cancer cells in animals. Compared to the widely ...

A Mouse Model of Fatigue Induced by Peripheral Irradiation

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. Here we describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. With appropriate shielding, the irradiation targets the lower abdominal/pelvic region of the mouse, sparing the brain, in an effort to model radiation treatment received by individuals with pelvic cancers. We deliver an irradiation dose that is sufficient to induce fatigue-like behavior in mice, measured by voluntary wheel-running activity (VWRA), without causing obvious morbidity. Since wheel running is a normal, voluntary behavior in mice, its use should have little confounding effect on other behavioral tests or biological measures. Hence, wheel running can be used as a feasible outcome measure in understanding the behavioral and biological correlates of fatigue. CRF is a complex condition with frequent comorbidities, and likely has causes related both to cancer and its various treatments. The methods described in this paper are useful for investigating radiation-induced changes that contribute to the development of CRF and, more generally, to explore the biological networks that can explain the development and persistence of a peripherally-triggered but centrally-driven behavior like fatigue.

We describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. The selected non-lethal irradiation dose leads to a week-long reduction in voluntary wheel-running activity.

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

Modeling Osteosarcoma Using Li-Fraumeni Syndrome Patient-derived Induced Pluripotent Stem Cells

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including osteosarcoma--one of the most frequent primary non-hematologic malignancies in the childhood and adolescence. Therefore, LFS provides an ideal model to study this malignancy. Taking advantage of iPSC methodologies, LFS-associated osteosarcoma can be successfully modeled by differentiating LFS patient iPSCs to mesenchymal stem cells (MSCs), and then to osteoblasts--the cells of origin for osteosarcomas. These LFS osteoblasts recapitulate oncogenic properties of osteosarcoma, providing an attractive model system for delineating the pathogenesis of osteosarcoma. This manuscript demonstrates a protocol for the generation of iPSCs from LFS patient fibroblasts, differentiation of iPSCs to MSCs, differentiation of MSCs to osteoblasts, and in vivo tumorigenesis using LFS osteoblasts. This iPSC disease model can be extended to identify potential biomarkers or therapeutic targets for LFS-associated osteosarcoma.

Here, we present a protocol for the generation of induced pluripotent Stem Cells (iPSCs) from Li-Fraumeni Syndrome (LFS) patient derived fibroblasts, differentiation of iPSCs via mesenchymal stem cells (MSCs) to osteoblasts, and modeling in vivo tumorigenesis using LFS patient-derived osteoblasts.

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including ...

Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, a small population of cells does not respond to TKI treatment, resulting in minimal residual disease (MRD); even the most potent TKIs fail to eradicate these cells. These MRD cells serve as a reservoir to develop resistance to therapy. Why TKI treatment is ineffective against MRD cells is not known. Growth factor signaling is implicated in supporting the survival of MRD cells during TKI treatment, but a mechanistic understanding is lacking. Recent studies demonstrated that an elevated c-Fos and Dusp1 expression as a result of convergent oncogenic and growth factor signaling in MRD cells mediate TKI resistance. The genetic and chemical inhibition of c-Fos and Dusp1 renders CML exquisitely sensitive to TKIs and cures CML in both genetic and humanized mouse models. We identified these target genes using multiple microarrays from TKI-sensitive and -resistant cells. Here, we provide methods for target validation using in vitro and in vivo mouse models. These methods can easily be applied to any target for genetic validation and therapeutic development.

Here, we describe protocols for the genetic and chemical validation of c-Fos and Dusp1 as a drug target in leukemia using in vitro and in vivo genetic and humanized mouse models. This method can be applied to any target for genetic validation and therapeutic development.

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, ...

A Human Peripheral Blood Mononuclear Cell (PBMC) Engrafted Humanized Xenograft Model for Translational Immuno-oncology (I-O) Research

The discovery and development of immuno-oncology (I-O) therapy in recent years represents a milestone in the treatment of cancer. However, treatment challenges persist. Robust and disease-relevant animal models are vital resources for continued preclinical research and development in order to address a range of additional immune checkpoints. Here, we describe a human peripheral blood mononuclear cell (PBMC) &#8212; based humanized xenograft model. BGB-A317 (Tislelizumab), an investigational humanized anti-PD-1 antibody in late-stage clinical development, is used as an example to discuss platform set-up, model characterization and drug efficacy evaluations. These humanized mice support the growth of most human tumors tested, thus allowing the assessment of I-O therapies in the context of both human immunity and human cancers. Once established, our model is comparatively time- and cost-effective, and usually yield highly reproducible results. We suggest that the protocol outlined in this article could serve as a general guideline for establishing mouse models reconstituted with human PBMC and tumors for I-O research.

A Human Peripheral Blood Mononuclear Cell PBMC Engrafted Humanized Xenograft Model for Translational Immuno-oncology I-O Research

We describe a human peripheral blood mononuclear cell (PBMC) &#8212; based humanized xenograft mouse model for translational immuno-oncology research. This protocol could serve as a general guideline for establishing and characterizing similar models for I-O therapy assessment.

The discovery and development of immuno-oncology (I-O) therapy in recent years represents a milestone in the treatment of cancer. However, treatment ...

In Vitro Assay to Study Tumor-macrophage Interaction

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a malignant brain tumor with no cure, has up to a half the tumor mass TAMs. TAMs can be pro-tumoral or anti-tumoral, depending on the activation of specific genes in the cells. Genetic mutations in the tumors, through regulating cytokine expression, can affect recruitment of TAMs to the tumor microenvironment. Here, we describe a quantitative cell-based assay to assess macrophage recruitment by the conditioned medium from the tumor cells. This assay uses the human macrophage cell line MV-4-11 to study macrophage attraction by the conditioned medium from glioblastoma, allowing for high reproducibility and low variability. Data generated with this assay can contribute to a better understanding of the interaction between the tumor and the tumor microenvironment. Similar assay can be used to assess interaction between the tumor cells and other immune cells, including T cells and natural killer (NK) cells.

This article represents a useful in vitro assay to evaluate the capability of conditioned medium from tumor cells to attract macrophages.

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

An Automated Differential Nuclear Staining Assay for Accurate Determination of Mitocan Cytotoxicity

The contribution of mitochondria to oncogenic transformation is a subject of wide interest and active study. As the field of cancer metabolism becomes more complex, the goal of targeting mitochondria using various compounds that inflict mitochondrial damage (so-called mitocans) is becoming quite popular. Unfortunately, many existing cytotoxicity assays, such as those based on tetrazolium salts or resazurin require functional mitochondrial enzymes for their performance. The damage inflicted by compounds that target mitochondria often compromises the accuracy of these assays. Here, we describe a modified protocol based on differential staining with two fluorescent dyes, one of which is cell-permeant (Hoechst 33342) and the other of which is not (propidium iodide). The difference in staining allows living and dead cells to be discriminated. The assay is amenable to automated microscopy and image analysis, which increases throughput and reduces bias. This also allows the assay to be used in high-throughput fashion using 96-well plates, making it a viable option for drug discovery efforts, particularly when the drugs in question have some level of mitotoxicity. Importantly, results obtained by Hoechst/PI staining assay show increased consistency, both with trypan blue exclusion results and between biological replicates when the assay is compared to other methods.

The protocol describes a rapid, high-throughput, reliable, inexpensive, and unbiased assay for efficiently determining cellular viability. This assay is particularly useful when cells&#39; mitochondria have been damaged, which interferes with other assays. The assay uses automated counting of cells stained with two nuclear dyes &#8211; Hoechst 33342 and propidium iodide.

The contribution of mitochondria to oncogenic transformation is a subject of wide interest and active study. As the field of cancer metabolism becomes ...

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

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage response induced by neutron-gamma mixed-beam used in boron neutron capture therapy (BNCT). Specifically, the proposed methodology is applied for the detection of repair proteins activation which can be visualized as foci using antibodies specific to DNA double-strand breaks (DNA-DSBs). DNA repair foci were assessed by immunofluorescence in colon cancer cells (HCT-116) after irradiation with the neutron-mixed beam. DNA-DSBs are the most genotoxic lesions and are repaired in mammalian cells by two major pathways: non-homologous end-joining pathway (NHEJ) and homologous recombination repair (HRR). The frequencies of foci, immunochemically stained, for commonly used markers in radiobiology like &#947;-H2AX, 53BP1 are associated with DNA-DSB number and are considered as efficient and sensitive markers for monitoring the induction and repair of DNA-DSBs. It was established that &#947;-H2AX foci attract repair proteins, leading to a higher concentration of repair factors near a DSB. To monitor DNA damage at the cellular level, immunofluorescence analysis for the presence of DNA-PKcs representative repair protein foci from the NHEJ pathway and Rad52 from the HRR pathway was planned. We have developed and introduced a reliable immunofluorescence staining protocol for the detection of radiation-induced DNA damage response with antibodies specific for repair factors from NHEJ and HRR pathways and observed radiation-induced foci (RIF). The proposed methodology can be used for investigating repair protein that is highly activated in the case of neutron-mixed beam radiation, thereby indicating the dominance of the repair pathway.

Radiation-induced DNA damage response activated by neutron mixed-beam used in boron neutron capture therapy (BNCT) has not been fully established. This protocol provides a step-by-step procedure to detect radiation-induced foci (RIF) of repair proteins by immunofluorescence staining in human colon cancer cell lines after irradiation with the neutron-mixed beam.

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

Isolating Human Peripheral Blood Mononuclear Cells and CD4+ T cells from S&#233;zary Syndrome Patients for Transcriptomic Profiling

Cutaneous T-cell lymphomas (CTCL) are derived from the transformation and uncontrolled proliferation of mature skin-homing T cells, and mycosis fungoides (MF) and S&#233;zary syndrome (SS) represent the most common subtypes. Despite a number of studies on characterizing gene expression, genetic alterations, and epigenetic abnormalities of CTCL, the molecular pathogenesis of MF/SS remains unclear. MF refers to the more common CTCL with a skin-predominance, and is usually limited to skin, whereas&#160;SS is an aggressive leukemic variant of CTCL with widespread skin involvement and is characterized by neoplastic distribution mainly involving blood, skin, and lymph node. Focusing on clinical practice, the identification of gene expression biomarkers has&#160;enormous potential to improve diagnosis and treatment of MF/SS. Indeed, recent transcriptomic studies have identified potential diagnostic biomarkers from differences in gene expression between normal and malignant T cells, which may improve our understanding of SS biology, and reveal potential therapeutic targets. This manuscript describes a detailed reproducible protocol for the isolation of peripheral blood mononuclear cells from fresh whole blood from patients diagnosed with SS, selection of CD4+ memory T cells (CD4+CD45RO+ T cells), chemical stimulation, and preparation of RNA suitable for transcriptomic profiling to discover novel prognostic molecular markers to gain additional insight in disease etiology. The stimulation using chemical agonist to activate nuclear regulation provides more specific assessment for pathways important in the dynamic transcription regulation and gene expression and eliminates confounding defects that may arise from upstream signaling defects arising from TCR antigen loss at the cell membrane. The data obtained from comparison of transcriptome of unstimulated to stimulated SS T cells unmasks functional regulatory gene expression defects not evident from analysis of quiescent unstimulated cells. Furthermore, the method outlined from this approach can be adapted for studying T cell gene expression defects in other T cell immune diseases.

We present a simple protocol for the isolation of peripheral blood mononuclear cells from whole blood obtained from patients diagnosed with S&#233;zary Syndrome, followed by selection of CD4+ T cells, their stimulation with phorbol12-myristate13-acetate and A23187 ionophore, and preparation of RNA for transcriptomic profiling.

Cutaneous T-cell lymphomas (CTCL) are derived from the transformation and uncontrolled proliferation of mature skin-homing T cells, and mycosis fungoides ...

Mapping the Structure-Function Relationships of Disordered Oncogenic Transcription Factors Using Transcriptomic Analysis

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these proteins contain an intrinsically disordered domain (IDD) fused with the DNA-binding domain (DBD) of another protein and orchestrate widespread transcriptional changes to promote malignancy. These fusions are often the sole recurring genomic aberration in the cancers they cause, making them attractive therapeutic targets. However, targeting oncogenic transcription factors requires a better understanding of the mechanistic role that low-complexity, IDDs play in their function. The N-terminal domain of EWSR1 is an IDD involved in a variety of oncogenic fusion transcription factors, including EWS/FLI, EWS/ATF, and EWS/WT1. Here, we use RNA-sequencing to investigate the structural features of the EWS domain important for transcriptional function of EWS/FLI in Ewing sarcoma. First shRNA-mediated depletion of the endogenous fusion from Ewing sarcoma cells paired with ectopic expression of a variety of EWS-mutant constructs is performed. Then RNA-sequencing is used to analyze the transcriptomes of cells expressing these constructs to characterize the functional deficits associated with mutations in the EWS domain. By integrating the transcriptomic analyses with previously published information about EWS/FLI DNA binding motifs, and genomic localization, as well as functional assays for transforming ability, we were able to identify structural features of EWS/FLI important for oncogenesis and define a novel set of EWS/FLI target genes critical for Ewing sarcoma. This paper demonstrates the use of RNA-sequencing as a method to map the structure-function relationship of the intrinsically disordered domain of oncogenic transcription factors.

Intrinsically disordered domains are important for oncogenic fusion transcription factor function. To therapeutically target these proteins, a more detailed understanding of the regulatory mechanisms employed by these domains is required. Here, we use transcriptomics to map important structural features of the intrinsically disordered EWS domain in Ewing sarcoma.

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these ...