1. Comet Assay

1. Reagent preparation
   1. For low-melting-point agarose, place 500 mg of low-melting agarose in 100 mL of DNA-/RNA-free water. Heat the bottle in a microwave oven until the agarose dissolves. Place the bottle in a 37 °C water bath until needed.
      NOTE: For further reading, see Azqueta et al., who studied the effects of agarose concentration on the DNA-unwinding time and several electrophoresis factors²³.
   2. Dilute SYBR green DNA dye in Tris-EDTA (TE) Buffer at a 1:10,000 ratio to obtain a 1x working stock dye solution. This dye is light-sensitive, so store it in a dark place.
   3. For the cell lysis buffer, dissolve 100 mL of lysis buffer (2.5 M NaCl, 0.1 M EDTA, 10 mM Tris, 1% Triton X-100) in deionized water (DI H₂O). Then, add 10 M NaOH to adjust the pH to 10.0. Cool the buffer to 4 °C.
   4. For the alkaline DNA-unwinding/electrophoresis running buffer, dissolve 1 L of alkaline solution (0.3 M NaOH, 1 mM EDTA) in DI H₂O. Cool the buffer to 4 °C. Make sure the pH >13.
   5. For precoated comet slides, place a few drops of 0.5% agarose on a glass slide. Immediately, using the flat surface of another slide, spread the agarose to form a thin layer of agarose coating.
      NOTE: Dry the slides before using.
   6. For iPS cell culture, briefly culture iPS cells in basement-membrane-matrix-coated (see Table of Materials) culture plates in mTESR1 culture medium until confluency is reached.
      NOTE: Human-cardiac-fibroblast-derived iPS cells were used in this protocol. The reprogramming of iPS cell derivation and culture conditions is described in recent articles from our research group^12,13,21.
2. Sample preparation
   1. Discard the culture medium and wash the cells. Dissociate the cells using detachment solution (see Table of Materials). Wash the cell pellet with phosphate-buffered saline (PBS) and resuspend the cells in PBS at 1 x 10⁵ cells/mL.
3. Sample slide preparation
   NOTE: Perform the following steps under low-light conditions to avoid ultraviolet-light-induced cell damage.
   1. Mix 10 μL of cell suspension and 90 μL of agarose solution (1:10 ratio) in a tube. Place the tube in a 37 °C water bath to prevent the agarose from solidifying.
   2. Mix the solutions without introducing air bubbles and pipette 70 μL/spot onto the precoated comet slide. Using a pipette tip, spread the cell agarose mixture to form a thin layer. Place the slide in 4 °C for 15 min.
   3. In a container, completely submerge the slide in cold lysis buffer. Place the container in the dark at 4 °C for 1 h.
   4. Replace the lysis buffer with cold alkaline solution. Make sure the slides are completely submerged. Place the container in the dark at 4 °C for 30 min.
4. Electrophoresis
   1. Place the slide in a horizontal electrophoresis tank. Fill the tank with cold alkaline electrophoresis buffer until the slides are completely submerged. Apply voltage at 1 V/cm for 15 to 30 min.
      NOTE: Total voltage = distance between the electrodes x 1 V
   2. In a container, completely submerge the slide in cold DI H₂O. After 2 min, remove the DI H₂O and add fresh DI H₂O. Repeat this step.
   3. Replace the DI H₂O with cold 70% ethanol. Wait 5 min. Gently remove the slide, do not tilt it, and let it air-dry.
   4. Once dried, add 100 μL of diluted DNA dye to each spot. Wait 15 min at room temperature. Using a FITC filter in an epifluorescence microscope, take images of 50 to 100 comets in total per sample.
5. Image analysis and calculation of the results
   1. For scoring the comets, use the National Institutes of Health’s (NIH) ImageJ with comet assay plugin (download ImageJ here: https://imagej.nih.gov/ij/download.html; download the plugin here: https://www.med.unc.edu/microscopy/resources/imagej-plugins-and-macros/comet-assay/).
      NOTE: The plugin comes with a PDF instruction which contains all details to perform the analysis. Additionally, screenshots of the comet analysis are shown in Figure 2A-C. Representative comet images of control and doxorubicin (Doxo)-treated iPS cells and the quantification of the comets are shown in Figure 3A-C.

2. DNA Damage Response

1. Sample and reagent preparation
   1. For an iPS-CM cell culture, culture iPS-CM cells in basement-membrane-matrix-coated (see Table of Materials) culture plates in RPMI medium supplemented with B-27 (CMM) until confluency is reached.
      NOTE: Human-iPS-cell-derived cardiomyocytes were used in this protocol. The protocol for iPS cell differentiation into cardiomyocytes can be found in recent articles from our research group^12,13,21.
   2. Seed iPS-CMs in chamber slides.
      1. Discard the culture medium from the iPS-CM wells. Wash the cells three times with PBS.
      2. Add 1 mL of 0.25% trypsin per well and incubate at 37 °C for 5 min. Check the plate under a microscope for cell detachment. Add 1 mL of CMM to stop the trypsin.
      3. Place cell suspension in a 15 mL tube and centrifuge for 5 min at 4 °C and 200 x g. Discard the medium, add 1 mL of CMM, and resuspend the cells. Count the cells and adjust the cell count to obtain 20 x 10⁵ cells in 1.6 mL of CMM.
      4. Seed 200 µL of cell suspension per well of the 8-well chamber slide (see Table of Materials). Tap the slide gently to spread the cells around the well and incubate at 37 °C.
   3. For the iPS-CM doxorubicin treatment, discard the culture medium from the iPS-CM wells. Wash the cells with PBS. Treat the cells with 1 μM doxorubicin and CMM for 4 h. Aspirate the medium and wash the cells with PBS.
   4. For the blocking buffer, prepare 10 mL of bovine serum albumin (BSA) solution containing 3% BSA and 0.05% Triton X-100. To prepare 10 mL of blocking buffer, add 1 mL of normal donkey serum to 9 mL of prepared BSA solution.
   5. For the primary antibody solution, add 1 μL of anti-rabbit phospho-histone H2A.X (Ser139) antibody to 200 μL of blocking buffer.
   6. For the secondary antibody mix, add 1 μL each of fluorochrome-conjugated anti-rabbit secondary antibody, DAPI, and fluorochrome-conjugated phalloidin to 200 μL of blocking buffer.
2. Immunolabeling
   1. Wash the cells three times with PBS and add 200 μL of freshly prepared 4% paraformaldehyde (PFA) to each well. Fix the cells for 20 min in the dark at room temperature. Wash the cells with PBS.
   2. Remove the PBS and add 200 μL of blocking buffer per well and block for 30 min in a humidified chamber at room temperature. Remove the blocking buffer and add 200 μL of primary antibody solution. Incubate for 45 min in a dark, humidified chamber at room temperature. Then, wash the wells three times with PBS.
   3. Remove the PBS and add 200 μL of secondary antibody mix. Incubate for 45 min in a dark, humidified chamber at room temperature. Then, wash the wells three times with PBS. Wash with DI H₂O before mounting them with antifade. Place a coverglass and store the slides in the dark at 4 °C.
   4. Using an epifluorescence microscope, take three to five images of random fields per sample, as shown in Figure 4A, and proceed to image analysis.
3. Counting of the DDR foci
   1. Download and install CellProfiler²⁴ (http://cellprofiler.org).
   2. NOTE: Image processing in this study was performed using CellProfiler version 2.1.1. Tutorials for CellProfiler that were used can be found elsewhere (https://www.youtube.com/watch?v=PvhRL9xpduk&list=PL7CC87670239B4D10). If a newer version of CellProfiler is being used, the pipeline might require minor adaptations.
   3. Download the pipeline punctae_gH2AX.cpipe. Select File > Import > Pipeline from the file and choose punctae_gH2AX.cpipe.
   4. Drag-and-drop the folder containing the acquired images of γH2A.X foci to the File list window in the Input modules/Images section for analysis (Figure 5A). Then, follow the instructions as shown in Figure 5A-L.
   5. Set the following parameters for the images:
      1. Drag the desired image for analysis onto the File list. Under Input modules, select Images (see Figure 5A for module settings). In Input modules, select Metadata (see Figure 5B for module settings). In Input modules, select NamesAndTypes (see Figure 5C for module settings).
      2. For Groups, select no. In Analysis modules, select ColorToGray (see Figure 5D for module settings).
   6. In Analysis modules, select Smooth (see Figure 5E for module settings).
      NOTE: This value may need to be optimized, depending on the image resolution.
   7. In Analysis modules, select IdentifyPrimaryObjects (see Figure 5F for module settings).
      NOTE: Optimize these values to make sure the whole nucleus is selected. After this step, the computer may lag.
   8. In Analysis modules, select Smooth (see Figure 5G for module settings).
      NOTE: This value may need to be optimized, depending on the image resolution.
   9. In Analysis modules, select EnhanceOrSuppressFeatures (see Figure 5H for module settings).
   10. In Analysis modules, select IdentifyPrimaryObjects (see Figure 5I for module settings).
       NOTE: Optimize these values to make sure the punctae are selected. After this step, the computer may lag again.
       1. In Analysis modules, select RelateObjects (see Figure 5J for module settings). In Analysis modules, select ClassifyObjects (see Figure 5K for module settings). In Analysis modules, select ExportToSpreadsheet (see Figure 5L for module settings).
       2. Click Analyze Images at the bottom left panel to perform image analysis. At the successful completion of the analysis, several images are generated (Figure 6A-F).
          NOTE: Users should save these images for future reference.
       3. Note the .csv file containing the quantitative data for further analysis that is generated and saved in the appropriate location on the computer. In the spreadsheet called MyExpt_Image, columns B and D will have the number of nuclei that have up to five and more punctae, respectively. Add columns B and D to get the total number of nuclei.
   11. Repeat step 2.3.3 – 2.3.8 for all images (change the MyExpt_ filename before every analysis). Plots can be made for assessing DNA integrity, as shown in Figure 4C.