Name: Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins (Video) | JoVE Journal
Uploaded: 2012-09-28T12:00:00.000+00:00
Duration: 10 min 24 s
Description: Learn two- and three-dimensional live cell imaging to visualize DNA damage response proteins like 53BP1 in real time. This method reveals spatiotemporal dynamics during mitosis and DNA repair.

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Supported in part by R01NS064593 and R21ES016636 (K.V.). Microscopy was performed at the VCU - Department of Neurobiology &amp; Anatomy Microscopy Facility, supported, in part, with funding from NIH-NINDS Center core grant 5P30NS047463. The spinning disc confocal microscope was purchased with an NIH-NCRR award (1S10RR027957).

A. Cell Preparation
<ol>
 <li>Normal human primary fibroblasts (GM02270) were obtained from Coriell Cell Repository, Camden, New Jersey, and immortalized with hTERT 6. Cells were grown and expanded in CellStar 6-cm dishes in media (4 ml) consisting of MEM supplemented with 20% fetal bovine serum (GIBCO), non-essential/essential amino acids, vitamins, sodium pyruvate, and penicillin/streptomycin (HyClone).</li>
 <li>Human embryonic kidney 293 (HEK293) cells were obtained from American Type Culture Collection and grown and expanded in CellStar 6-cm dishes. The cells were kept in media (4 ml) consisting of DMEM supplemented with 10% fetal bovine serum, non-essential amino acids, L-glutamine, and penicillin/streptomycin.</li>
 <li>53BP1-mCherry (N-Myc-53BP1 WT pLPC-Puro; Addgene plasmid 19836) and H2B-GFP (pCLNR-H2BG; Addgene plasmid 17735) fusion gene constructs also expressing puromycin or G418 resistance genes, respectively, were transduced (fibroblasts) or transfected (HEK293) using SuperFect (Qiagene) into cells and maintained under drug selection. Maintenance of fluorescence was periodically checked.</li>
 <li>24-48 hr before image acquisition, cells were trypsinized into a single-cell suspension and seeded at a low density onto 3.5-cm FluroDish glass bottom plates.</li>
</ol>
B. Microscope Setup and Image Acquisition
This protocol was developed using the Zeiss Cell Observer SD spinning disk confocal microscope equipped with an AxioObserver Z1 stand, a dual-channel Yokagawa CSU-X1A 5000 spinning disk unit, 2 Photometrics QuantEM 512SC emCCD cameras, an HXP 120C fiber-based illuminator, 4 lasers (a Lasos 100mW multi-line Argon [458, 488, 514 nm], 50 mW 405 nm diode, 40 mW 561 nm diode, and 30 mW 635 nm diode), AOTF, a Pecon XL multiS1 stage incubation system, Zeiss incubation modules (O2 Module S, CO2 Module S, TempModule S, Heating Unit XL S) and a Prior motorized XY stage with a NanoScanZ piezo Z insert. To minimize spherical aberrations while imaging live cells supported in an aqueous medium, a C-Apochromat 63x/1.20 Water/Corr objective lens and Zeiss Immersol W immersion fluid (with a refractive index n = 1.334) were used. For 2 channel confocal imaging, a RQFT 405/488/568/647 dichroic mirror and BP525/50 (green) and BP629/62 (red) emission filters were used. The system software used was Zeiss Axiovision (ver. 4.8.2.0) with AV4 Multi-channel/Z/T, Fast Imaging, Physiology, MosaiX, Mark &amp; Find, Dual Camera, Inside 4D, Autofocus and 3-D Deconvolution modules.
<ol>
 <li>Ensure that CO2 gas is running to the CO2 Module of the incubation system. If the microscope is supported by an anti-vibration air table, turn on the air supply (or N2 gas) for the air table.</li>
 <li>Turn on the power for the microscope stand, spinning disk unit, cameras, incubation modules, HXP illuminator, motorized stage, Argon laser, and computer.</li>
 <li>After 1 min of warm-up time, turn the ignition key for the Argon laser to "On".</li>
 <li>On the Zeiss laser control panel, turn on the switches for the laser lines to be used.</li>
 <li>Switch the toggle switch for the Lasos Argon laser controller from "standby" to "laser run" and adjust the light controller to the optimal level (i.e. just below the point where the green indicator turns red).</li>
 <li>Start up the AxioVision software. Note: the user interface for AxioVision may be customized with windows and pull-down menus that are specific for the particular microscope and components that it controls. As such, each system has a potentially unique interface. Therefore, generic instructions for software manipulation are provided in subsequent steps, rather than directions for specific software windows, tabs and/or pull-down menus.</li>
 <li>Approximately 1 hr prior to imaging, in the software, locate the controls for the incubator and turn on the heating for the upper chamber and the stage plate. Set the temperature to 37 °C. Turn on the CO2 control and set the level at 5%.</li>
 <li>Select the objective lens for imaging. In this study, a 63x/1.20 N.A. C-Apochromat Water/Corr objective lens was used. Note: For this lens, an immersion medium with a refractive index similar to water (Zeiss Immersol W immersion fluid) is needed.</li>
 <li>If multiple separate positions are to be sampled over a long period of time, be certain to apply a sufficient amount of immersion medium to ensure that it is carried from one position to another.</li>
 <li>Place the dish on the stage and bring the objective lens up into contact with the bottom.</li>
 <li>Using either the microscope controls or the software, direct the emitted light to the eyepieces and select the appropriate widefield filter set for the fluorescent signal of interest (for this study, "Red" filter set for 53BP1 and a "GFP" filter set for H2B).</li>
 <li>View through the ocular lenses, focus the image, and locate a suitable field of cells.</li>
 <li>Using either the software or the microscope controls, direct the emitted light away from the eyepieces to the port with the confocal spinning disc unit.</li>
 <li>In the software, turn on the appropriate laser (for this study, the 561 nm laser for 53BP1 and the 488 nm line of the Argon laser for H2B).</li>
 <li>For each channel, adjust the intensity of the laser by adjusting the acousto-optic tunable filter (AOTF) control to an appropriate level.</li>
 <li>Select the appropriate dichroic mirror (RQFT 405/488/568/647) and emission filters (BP 629/62 for 53BP1 and BP 525/50 for H2B). Open the shutter to the spinning disc unit.</li>
 <li>Select the "Live" window to display the current field of view.</li>
 <li>In the software, open the "Camera" control, select the camera to be used (if a dual camera system) and set exposure time to approximately 100 ms. Adjust the % and EM gain as necessary. Note: The 53BP1-mCherry construct appears somewhat dim. We found it helpful to increase the EM gain.</li>
 <li>In the software, open the control for the confocal spinning disc unit and adjust the spinning disc speed by entering the camera exposure time that was set to capture a suitable image (e.g. ~100 ms). Click "Set" to lock in the change.</li>
 <li>Open "multi-dimensional acquisition". Select the channel tab and load/select appropriate channels. For this study, we're using channels defined for dsRed (561 nm laser excitation and BP 629/62 filter for emission) and GFP (488 nm laser line for excitation and BP525/50 filter for emission).</li>
 <li>To ensure image registration, a common dichroic mirror (RQFT 405/488/568/647) was used for both channels. Set the software to "Autofocus". Note: Go to Tools→ Settings editor to adjust the MDA settings. Ensure that adequate laser power is set ("Adequate laser power" refers to a setting which enables you to excite the appropriate fluorophore while minimizing photo-bleaching).</li>
 <li>In the MDA window, select the z-stack tab. Select "Z-stack at current focus position". Set the range for ~10 μm z-stack and choose "optimal" for the number of steps to ensure Nyquist sampling through Z. Note: The exact size of the z-stack will depend on the height of your cells. Adjust accordingly.</li>
 <li>23. Click "Start" and analyze the resulting z-stack image to ensure the settings are appropriate for what you are investigating (e.g. in this study, it was important to image the entire nucleus).</li>
 <li>Select the "T" (time) tab. For our experiment with camptothecin, we set the interval between imaging timepoints to 5 min and the overall duration of the session for 1 hr for a control (non-treated cells) video. Note: to minimize photo-bleaching of the cells, the experiment should be configured such that the AOTF blanks the laser between imaging time points.</li>
 <li>For multi-point imaging of several cells in the dish, in the MDA window, select the position tab. Ensure "Apply 'setting before/after time point' per position" is checked. Select "Mark_Find". Using the "Live" view, move the dish around and select appropriate fields of view.</li>
 <li>Click "Start" in the multi-dimensional-acquisition menu to begin the experiment and record a control video (of untreated cells).</li>
 <li>After the control video, add the appropriate treatment (in this case, 10 μM camptothecin). We recorded the experimental (drug-treated cells) video at 5-10 min intervals for 2-4 hr. Note: An important issue to consider is the speed with which a given effect occurs after treatment. As seen in the video, 53BP1 foci begin to from ~5 min after addition of CPT. Though a relatively easy process, adding drug to the dish manually and re-calibrating the microscope does take time. Consideration must be given to this when designing experiments.</li>
 <li>For monitoring mitosis, we set the interval to 7.5 min and recorded for 4-5 hr (the specific settings depend upon the cell line used and the length of its cell cycle). Adjustments may need to be made to prevent photo-bleaching during longer recordings.</li>
</ol>
C. Image Processing and Analysis
This protocol was developed using Volocity software (PerkinElmer). The software used to acquire this data (AxioVision) also has the ability to process and analyze images. Users are encouraged to utilize the software available to them, and consult appropriate literature regarding their application.
<ol>
 <li>Open Volocity software. Create and name a new library, and import your video files.</li>
 <li>For viewing the files, we usually find it most helpful to use the "Extended Focus" setting. This superimposes the z-stack slices and, for this protocol, allows us to visualize 53BP1 foci in different areas of the nucleus.</li>
 <li>Adjust videos as necessary. Volocity is equipped with a range of tools to improve the quality of acquired images. By ensuring the settings on the microscope were appropriate, much time can be saved in editing later on. Often, it is helpful to deconvolve your images, and adjust the brightness/contrast. Your specific editing needs will vary based on the experiment.</li>
 <li>Add a relative time stamp and scale bar.</li>
 <li>To view cells in 3-D, switch to the "3-D Opacity" setting. This permits rotation of the 3-D rendered cells in space, thus providing multiple perspectives of structures of interest within the cells. Note: It is helpful to visualize cells in different planes to determine where structures of interest travel. For example, in the "Extended Focus" setting it is difficult to discern that the 53BP1 does, in fact, dissociate from DNA during mitosis. However, this is readily apparent in 3-D.</li>
 <li>Movies and still images may be exported into a variety of file types based on user preference.</li>
</ol>
D. Representative Results
An example of 53BP1 foci formation in response to CPT is shown in Figure 1. Cells exposed to CPT form foci within 5-10 min, and maintain these foci throughout the duration of recording. As shown in Figure 2, 53BP1 dissociates from chromatin at the onset of mitosis, forming a thin haze around the condensing chromosomes. As telophase occurs and mitosis comes to an end, 53BP1 once again aggregates into distinct foci. While the HEK293 cells were not exposed to CPT, they nevertheless formed abundant, spontaneous 53BP1 repair foci generated by endogenous DNA damage. This observation allowed us to conclude that 53BP1 does not form foci during early mitosis, in line with a previous report showing a similar effect after the exposure of cells to ionizing radiation and radiomimetic drugs 5.
<img alt="Fluorescence microscopy image of cell nuclei with and without Camptothecin treatment, 21 μm scale." src="/files/ftp_upload/4251/4251fig1.jpg" data-altupdatedat="1787630408000" data-alt="Figure 1"> 
 Figure 1. Camptothecin (10 μM) causes DSBs and 53BP1 foci formation in cycling fibroblasts within 30 min of adding the drug to the medium.
<img alt="Cell division phases, prometaphase to telophase, fluorescence microscopy, mitosis, cell-cycle analysis." src="/files/ftp_upload/4251/4251fig2.jpg" data-altupdatedat="1787630408000" data-alt="Figure 2"> 
 Figure 2. 53BP1 does not form foci during mitosis until telophase/G1 in HEK293 cells.

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No conflicts of interest declared.

Maintenance of genomic integrity is crucial for cell survival. Failure to preserve the genome results in premature aging, carcinogenesis, or death 8. There is intense interest in discerning how the DDR functions, stemming from its importance to both basic and clinical research. Many techniques have been developed over the years to aid in the study of how cells detect and repair DNA damage. Traditional methods such as immunocytochemistry and western blotting have been mainstays of the field, though recen...

<table><tbody><tr><td>CellStar culture dishes</td><td>Greiner Bio-one</td><td></td><td></td></tr><tr><td>FluroDish glass bottom dishes</td><td>World Precision Instruments, Inc.</td><td></td><td></td></tr><tr><td>MEM media</td><td>GIBCO</td><td></td><td></td></tr><tr><td>Non-essential amino acids</td><td>GIBCO</td><td></td><td></td></tr><tr><td>Amino acids</td><td>GIBCO</td><td></td><td></td></tr><tr><td>Vitamins</td><td>GIBCO</td><td></td><td></td></tr><tr><td>Sodium Pyruvate</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>Penicillin/Streptomycin </td><td>HyClone</td><td></td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>GIBCO</td><td></td><td></td></tr><tr><td>N-Myc-53BP1 WT pLPC-Puro;&lt;br/&gt; plasmid 19836</td><td>Addgene</td><td></td><td></td></tr><tr><td>pCLNR-H2BG; plasmid 17735</td><td>Addgene</td><td></td><td></td></tr><tr><td>SuperFect</td><td>Qiagen</td><td></td><td></td></tr><tr><td>Zeiss Cell Observer SD Imaging system</td><td>Zeiss</td><td></td><td></td></tr><tr><td>AxioVision (release 4.8.2)</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Zeiss Immersol W Oil</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Volocity software (version 6.0)</td><td>PerkinElmer</td><td></td><td></td></tr></tbody></table>

two- and three-dimensional live cell imaging of dna damage response proteins

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

This demonstration uses two and 3D live cell imaging to understand the spatial temporal relationships of proteins involved in the DNA damage response. First, transfect or transduce your cell line of choice with expression constructs containing appropriate fluorescent markers. Here M Cherry and GFP next acquire a series of control videos showing the fluorescently labeled cells under normal conditions.Then apply an appropriate treatment and capture data of the treatment effects on the fluorescent cells process and analyze the video data for spatial temporal relationships of proteins. Ultimately, live cell fluorescence microscopy can detail cellular dynamics like the formation of 53 BP one foci in response to DNA damaging agents or 53 BP one's behavior during mitosis. We first became interested in two and 3D lifestyle imaging when we wanted to study how varying treatments affected mitosis in certain cell lines.This experimental approach allows us to study the DNA damage response and the maintenance of genomic stability more easily than traditional methods. Additionally, you can use this method to study how certain proteins interact in various signaling pathways and various cell lines. The main advantage of lifestyle imaging over traditional techniques like cyto chemistry is that it allows the study of the DNA damage response proteins in real time.Optimizing the right recording conditions can be challenging. This demonstration can be used to see the many small steps needed to ensure success. Remember that persistence pays off when it comes to minimizing photo bleaching, finding the right recording intervals and the right recording length of time, et cetera.Transfect or transduce the appropriate cell lines with the plasmids for mCherry and GFP fusion protein expression. Maintain the cultures under drug selection and periodically check for expression of the fluorescent proteins the day before image acquisition. Harvest the cells using trypsin.Then seed low density cultures onto 3.5 centimeter fluoro dish glass bottom plates. This protocol was developed using the Zeiss cell observer SD spinning disc confocal microscope, equipped with an axio observer Z one stand First, verify that the CO2 gas is running to the CO2 module of the incubation system. If the microscope is supported by an anti vibration Airtable, then turn on the air supply for the Airtable.Now switch on the power for the microscope stand spinning disc unit cameras, incubation modules, HXP illuminator, motorized stage, argonne laser, and computer. Turn on the switches for the laser lines to be used. Start up the Axio vision software.The Avision software may be customized with windows and pull down menus that are specific to the microscope and components that it controls. As such, each user interface has a potentially unique look Approximately one hour prior to imaging. Locate the controls for the incubator and turn on the heating for the upper chamber and the stage plate.Set the temperature to 37 degrees Celsius. Turn on the CO2 control and set the level at 5%Select the objective lens for imaging In this study. The objective lens requires use of an immersion medium that has a refractive index similar to water.If you need to image multiple positions over long periods of time, be sure to apply enough immersion medium so that the scope does not dry out. Place the culture dish on the stage and bring the objective lens up into contact with the bottom, using either the microscope controls or the software. Direct the emitted light to the eye pieces and select the appropriate wide field filter set for the fluorescent signal of interest.Now view through the ocular lenses. Focus the image and locate a suitable field of cells using either the software or the microscope controls. Direct the emitted light away from the eye pieces to the port with the confocal spinning disc unit in the software.Turn on the appropriate laser for this study. For each channel, adjust the intensity of the laser by adjusting the kuo optic tunable filter control to an appropriate level. Next, select the appropriate dichroic mirror and emission filters.Open the shutter to the spinning disc unit. Now select the live window to display the current field of view. Now open the camera control window.Select the camera to be used and set exposure time to approximately 100 milliseconds. Adjust the percent and EM gain as necessary. Also, open the control for the confocal spinning disc unit and adjust the spinning disc speed by entering the camera exposure time that was set to capture a suitable image.Click set to lock in the change. Now open the multi-dimensional acquisition window. Select the channel tab and load or select appropriate channels defined for the Flora Fours.To ensure image registration, use a common dichroic mirror for both channels. Set the software to auto focus. Proceed to the MDA window and click on the ZS stack tab.Select ZS stack at current focus position. Set the range for a 10 micron Zack and choose optimal for the number of steps to ensure nyquist sampling through Z.Now click start and analyze the resulting Z stack image. Select the time tab.Set the interval between imaging time points and the overall duration of the session. For multi-point imaging of several cells in the dish, open the MDA window. Select the position tab and verify that the apply setting before or after time point per position is checked.Now select mark. Find using the live view, move the dish around and select appropriate fields of view. Click start in the multidimensional acquisition menu to begin the experiment and record a control video.Proceed to add the appropriate treatment and record the experimental video at determined time intervals for a defined period of time. For this cell line and for monitoring the entire process of mitosis, we used an interval of seven and a half minutes and it for four to five hours. You will have to determine the particular settings for your cell line and for what you want to study.Open the velocity software, create and name a new library and import the experimental video files for viewing the files. Try using the extended focus setting, adjust the videos as necessary. Velocity is equipped with a range of tools to help improve the quality of your acquired images.If the settings on the microscope are appropriate, it will save a lot of time later on In editing. Often it is helpful to devolve your images and adjust the brightness and contrast your specific needs will vary based on your experiment To view the cells in 3D. Switch to the 3D opacity setting.This permits rotation of the 3D rendered cells in space, thus providing multiple perspectives of structures of interest within the cells.Movies. Still images can be exported into a variety of file types based on user preference Compared to the controls. Fibroblast cells exposed to CPT formed foci within five to 10 minutes and maintained these foci throughout the duration of recording.Interestingly, in human embryonic kidney cells, 53 BP one dissociates from chromatin at the onset of mitosis, forming a thin haze around the condensing chromosomes. As teleph occurs and mitosis comes to an end 53 BP one, once again aggregates into distinct foci. We hope that this video will enable you to study the spatiotemporal relationships of proteins involved in the DNA damage response as well as other pathways.For example, in the field of chromatin dynamics, this technique was used to study how 53 BP one binding affects the movement of telomeric ends of DNA. After the creation of genetically modified cells, two and 3D live cell imaging can be performed in approximately four to eight hours. Ensure that the microscope settings are adjusted appropriately to have optimal imaging.

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Research

JoVE Journal

Biology

In this video, we describe the immunofluorescence method to visualize the localization of the DNA repair proteins γH2AX and 53BP1 at the sites of double-strand DNA breaks in irradiated cell nuclei.

Immunofluorescence-Based Visualization of DNA Repair Protein Interactions

Lasers are frequently used in studies of the cellular response to DNA damage. However, they generate lesions whose spacing, frequency, and collisions with replication forks are rarely characterized. Here, we describe an approach that enables the determination of these parameters with laser localized interstrand crosslinks.

Single Molecule Analysis of Laser Localized Psoralen Adducts

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Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

Laser microirradiation is a useful tool for studies of DNA repair in living cells. A methodological approach for the use of UVA lasers to induce various DNA lesions is shown. We have optimized a method for local microirradiation that maintains the normal cell cycle; thus, irradiated cells proceed through mitosis.

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

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Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

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Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells

Studying DNA damage repair kinetics requires a system to induce lesions at defined sub-nuclear regions. We describe a method to create localized double-stranded breaks using a laser-scanning confocal microscope equipped with a 405 nm laser and provide automated procedures to quantify the dynamics of repair factors at these lesions.

Investigation of Protein Recruitment to DNA Lesions Using 405 Nm Laser Micro-irradiation

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

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

Following DNA damage, human cells activate essential repair pathways to restore the integrity of their genome. Here, we describe the method of indirect immunofluorescence as a means to detect DNA repair proteins, analyze their spatial and temporal recruitment, and help interrogate protein-protein interaction at the sites of DNA damage.

Visualization of DNA Repair Proteins Interaction by Immunofluorescence

This article introduces essential steps of immunostaining and chromatin immunoprecipitation. These protocols are commonly used to study DNA damage-related cellular processes and to visualize and quantify the recruitment of proteins implicated in DNA repair.

Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage

This protocol presents a new reporter gene system and the experimental setup to detect transcription at DNA double-strand breaks with single-molecule sensitivity.

Live-Cell Imaging of Transcriptional Activity at DNA Double-Strand Breaks

Learn two- and three-dimensional live cell imaging to visualize DNA damage response proteins like 53BP1 in real time. This method reveals spatiotemporal dynamics during mitosis and DNA repair.

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

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