We thank Mrs. Akiko Shibata for technical assistance. This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan for programs for Leading Graduate Schools, Cultivating Global Leaders in Heavy Ion Therapeutics and Engineering.

1. Preparation of Materials
<ol>
	<li>Autoclave cover slips.
	<ol>
		<li>Place cover slips in a glass beaker.</li>
		<li>Cover the glass beaker with foil.</li>
		<li>Autoclave at 121 &#176;C at 15 psi for 20 min.</li>
		<li>Dry at 50 &#176;C. Store at room temperature in a culture hood.</li>
	</ol>
	</li>
	<li>Prepare Fixation Solution: 3% paraformaldehyde + 2% sucrose in phosphate-buffered saline (PBS).
	<ol>
		<li>To a 1,000 mL glass bottle, add 700 mL PBS and 30 g paraformaldehyde. 
		CAUTION: Paraformaldehyde is corrosive, wear appropriate gloves.</li>
		<li>Dissolve paraformaldehyde completely by heating to 80 &#176;C using a microwave. 
		CAUTION: Paraformaldehyde fumes are toxic, wear a mask.</li>
		<li>Leave at room temperature overnight.</li>
		<li>Add 20 g sucrose.</li>
		<li>Add PBS to make the total volume 1 L.</li>
		<li>Aliquot in 50 mL tubes. Fixation Solution can be stored for 3 years at -20 &#176;C or for 3 months at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Preparation of Cell Culture
NOTE: These procedures should be performed in a culture hood.
<ol>
	<li>Culture and passage U2OS human osteosarcoma cells to maintain logarithmic growth5. 
	NOTE: Other cell types can be used after determining the optimal irradiation dose.</li>
	<li>Wipe a scalpel with paper towels moistened with 70% ethanol. Using the scalpel, place a cover slip on a 35 mm cell culture dish (hereafter simply referred to as &#34;the dish&#34;). 
	NOTE: The number of cover slips in one dish can be increased according to the experimental design (see Discussion).</li>
	<li>Add 1 mL culture media: DMEM supplemented with 10% fetal bovine serum. 
	NOTE: Other media can be used according to the chosen cell type.</li>
	<li>Wipe forceps with paper towels moistened with 70% ethanol. Using the forceps, gently hold the center of the cover slip. Aspirate the culture media from the dish completely, while maintaining a hold on the cover slip. 
	&#8203;NOTE: This step promotes immobilization of the cover slip to the dish.</li>
	<li>Detach adherent cultured cells using trypsin5.
	<ol>
		<li>Aspirate culture media from the dish.</li>
		<li>Add 1 mL PBS and shake the dish gently.</li>
		<li>Aspirate PBS from the dish.</li>
		<li>Add 1 mL trypsin [0.25w/v% Trypsin-1mmol/L EDTA] to the dish.</li>
		<li>Incubate for 5 min at 37 &#176;C in an atmosphere containing 5% CO2.</li>
		<li>Add 9 mL culture media to the dish.</li>
		<li>Using a 1000 &#956;L micropipette, prepare a single-cell suspension.</li>
	</ol>
	</li>
	<li>Count the cells5.
	<ol>
		<li>In a 1.5 mL tube, add 0.9 mL cell suspension and 0.1 mL 0.4% Trypan Blue solution.</li>
		<li>Apply 10 &#956;L to a hemocytometer.</li>
		<li>Examine the number of unstained (i.e., live) cells under an inverted-phase microscope.</li>
	</ol>
	</li>
	<li>In a 15 mL tube, prepare 3 mL cell suspension in culture media at a density of 0.5 x 105 cells/mL. 
	NOTE: Cell density can be modified according to experimental need (see Discussion).</li>
	<li>Gently add 2 mL of the cell suspension to the dish.</li>
	<li>Incubate overnight at 37 &#176;C in an atmosphere containing 5% CO2.</li>
</ol>
3. Irradiation
CAUTION: Handle the X-ray irradiator carefully according to the manufacturer&#39;s instructions.
<ol>
	<li>Examine the cells under an inverted-phase microscope. Confirm that the cells are attached to the cover slip and alive.</li>
	<li>Irradiate the dish with 6 Gy X-rays (1.4 Gy/min, 300 KVP, 20 mA).</li>
	<li>Incubate for 72 h at 37 &#176;C in an atmosphere containing 5% CO2. 
	NOTE: Incubation time can be modified according to the experimental design (see Discussion).</li>
</ol>
4. Fixation
<ol>
	<li>Aspirate culture media from the dish.</li>
	<li>Using scissors, cut the tip of a 1,000 &#956;L micropipette tip 5 mm from the end. 
	NOTE: The cut tip helps to smoothly apply the Fixation Solution.</li>
	<li>Using the cut tip, add 1 mL Fixation Solution (prepared in step 1.2) to the dish from the side wall of the dish. 
	NOTE: This step is time-sensitive and should therefore be performed without changing micropipette tips when handling multiple samples. Application of Fixation Solution directly to the bottom of the dish can damage the cells.</li>
	<li>Place the dish in a square culture dish. Shake the square culture dish gently to distribute the Fixation Solution over the cover slip evenly.</li>
	<li>Incubate for 10 min at room temperature.</li>
	<li>Aspirate Fixation Solution from the dish.</li>
	<li>Add 2 mL of PBS to the dish from the side wall of the dish. 
	NOTE: Application of PBS directly to the bottom of the dish can damage the cells.</li>
	<li>Aspirate PBS from the dish.</li>
	<li>Repeat steps 4.7 and 4.8 twice. 
	NOTE: The dish can be stored for 1 week at 4 &#176;C with the cover slips bathed in 2 mL PBS.</li>
</ol>
5. DAPI Staining
<ol>
	<li>To a slide glass, apply 5 &#956;L 1 &#956;g/mL DAPI staining reagent. 
	NOTE: The DAPI staining reagent is stable for 6 months when stored protected from light at or below -20 &#176;C.</li>
	<li>Using a scalpel, remove the cover slip from the dish.</li>
	<li>Draw off the excess PBS on the cover slip by touching the edge of the cover slip with a paper towel.</li>
	<li>Mount the cover slip upside-down onto the drop of DAPI staining reagent on the slide glass so that the cells are exposed to DAPI staining reagent. 
	NOTE: This step is time-sensitive. Drying of the DAPI staining reagent can lead to suboptimal staining.</li>
	<li>The samples can be stored for 1 year at -20 &#176;C.</li>
</ol>
6. Image Acquisition
<ol>
	<li>Examine the sample on a fluorescence microscope. Nuclei are visualized using the DAPI filter. 
	NOTE: Either a 20X or a 60X oil lens can be used, according to the researcher&#39;s interest. When using a 60X oil lens, add one drop of oil onto the cover slip. Be careful that the samples do not touch the lens; otherwise, the lens can be damaged.</li>
	<li>Acquire images of nuclei using a CCD camera and digital image acquisition software with the following settings and parameters: DAPI filter, monolayer images, gain of 1X, and automatic exposure.</li>
	<li>Finish image acquisition: Wipe the 20X lens with paper towels moistened with lens cleaner. Wipe the 60X oil lens with paper towels moistened with chloroform.</li>
</ol>
7. Evaluation of Clonogenic Cell Death Mode
<ol>
	<li>In randomly selected images, count the number of nuclei that meet the criteria for apoptosis, mitotic catastrophe, and cellular senescence until the total number of counted nuclei reaches 300. Conduct this step in triplicate for each experimental setting.
	<ol>
		<li>Criteria for apoptosis: presence of apoptotic bodies (i.e., condensed and fragmented nuclei)6,7.</li>
		<li>Criteria for mitotic catastrophe: presence of nuclei showing two or more distinct lobes or micronuclei8,9,10.</li>
		<li>Criteria for cellular senescence: presence of senescence-associated heterochromatic foci (SAHF) (i.e., nuclear DNA containing 30-50 bright, dense foci)2,11.</li>
	</ol>
	</li>
</ol>

<ol><li>Wouters, B. G. Cell death after irradiation: how, when and why cells die. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aWouters%2C+BG+%22Basic+Clinical+Radiobiology.+4th+ed%22">Basic Clinical Radiobiology. 4th ed</a>. Joiner, M. C., van der Kogel, A. J. , Hodder Education. London. 27-40 (2009).</li>
<li>Aird, K. M., Zhang, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detection+of+senescence-associated+heterochromatin+foci+%28SAHF%29%5BTitle%5D)+AND+%22Methods+Mol.+Biol%22%5BJournal%5D)">Detection of senescence-associated heterochromatin foci (SAHF).</a> Methods Mol. Biol. 965, 185-196 (2013).</li>
<li>Kobayashi, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitotic+catastrophe+is+a+putative+mechanism+underlying+the+weak+correlation+between+sensitivity+to+carbon+ions+and+cisplatin%5BTitle%5D)+AND+%22Sci.+Rep%22%5BJournal%5D)">Mitotic catastrophe is a putative mechanism underlying the weak correlation between sensitivity to carbon ions and cisplatin.</a> Sci. Rep. 7, 40588(2017).</li>
<li>Amornwichet, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Carbon-ion+beam+irradiation+kills+X-ray-resistant+p53-null+cancer+cells+by+inducing+mitotic+catastrophe%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Carbon-ion beam irradiation kills X-ray-resistant p53-null cancer cells by inducing mitotic catastrophe.</a> PLoS One. , 115121(2014).</li>
<li>Masters, J. R., Stacey, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Changing+medium+and+passaging+cell+lines%5BTitle%5D)+AND+%22Nat.+Protoc%22%5BJournal%5D)">Changing medium and passaging cell lines.</a> Nat. Protoc. 2, 2276-2284 (2007).</li>
<li>Sawai, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effectiveness+of+sulforaphane+as+a+radiosensitizer+for+murine+osteosarcoma+cells%5BTitle%5D)+AND+%22Oncol.+Rep%22%5BJournal%5D)">Effectiveness of sulforaphane as a radiosensitizer for murine osteosarcoma cells.</a> Oncol. Rep. 29, 941-945 (2013).</li>
<li>Cummings, B. S., Wills, L. P., Schnellmann, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Measurement+of+cell+death+in+mammalian+cells%5BTitle%5D)+AND+%22Curr.+Protoc.+Pharmacol%22%5BJournal%5D)">Measurement of cell death in mammalian cells.</a> Curr. Protoc. Pharmacol. , Suppl. 56, Chapter 12: Unit 12.8 (2012).</li>
<li>Oike, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((C646%2C+a+selective+small+molecule+inhibitor+of+histone+acetyltransferase+p300%2C+radiosensitizes+lung+cancer+cells+by+enhancing+mitotic+catastrophe%5BTitle%5D)+AND+%22Radiother.+Oncol%22%5BJournal%5D)">C646, a selective small molecule inhibitor of histone acetyltransferase p300, radiosensitizes lung cancer cells by enhancing mitotic catastrophe.</a> Radiother. Oncol. 111, 222-227 (2014).</li>
<li>Oike, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+synthetic+lethality-based+strategy+to+treat+cancers+harboring+a+genetic+deficiency+in+the+chromatin+remodeling+factor+BRG1%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">A synthetic lethality-based strategy to treat cancers harboring a genetic deficiency in the chromatin remodeling factor BRG1.</a> Cancer Res. 73, 5508-5518 (2013).</li>
<li>Russo, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+and+in+vivo+radiosensitization+of+glioblastoma+cells+by+the+poly+%28ADP-ribose%29+polymerase+inhibitor+E7016%5BTitle%5D)+AND+%22Clin.+Cancer+Res%22%5BJournal%5D)">In vitro and in vivo radiosensitization of glioblastoma cells by the poly (ADP-ribose) polymerase inhibitor E7016.</a> Clin. Cancer Res. 15, 607-612 (2009).</li>
<li>Di Micco, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interplay+between+oncogene-induced+DNA+damage+response+and+heterochromatin+in+senescence+and+cancer%5BTitle%5D)+AND+%22Nat.+Cell+Biol%22%5BJournal%5D)">Interplay between oncogene-induced DNA damage response and heterochromatin in senescence and cancer.</a> Nat. Cell Biol. 13, 292-302 (2011).</li>
<li>Franken, N. A., Rodermond, H. M., Stap, J., Haveman, J., van Bree, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clonogenic+assay+of+cells+in+vitro%5BTitle%5D)+AND+%22Nat.+Protoc%22%5BJournal%5D)">Clonogenic assay of cells in vitro.</a> Nat. Protoc. 1, 2315-2319 (2006).</li>
<li>Vakifahmetoglu, H., Olsson, M., Zhivotovsky, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Death+through+a+tragedy%3A+mitotic+catastrophe%5BTitle%5D)+AND+%22Cell+Death+Differ%22%5BJournal%5D)">Death through a tragedy: mitotic catastrophe.</a> Cell Death Differ. 15, 1153-1162 (2008).</li>
<li>Imreh, G., Norberg, H. V., Imreh, S., Zhivotovsky, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chromosomal+breaks+during+mitotic+catastrophe+trigger+%CE%B3H2AX-ATM-p53-mediated+apoptosis%5BTitle%5D)+AND+%22J.+Cell+Sci%22%5BJournal%5D)">Chromosomal breaks during mitotic catastrophe trigger γH2AX-ATM-p53-mediated apoptosis.</a> J. Cell Sci. 129, 1950(2016).</li>
<li>Torres-Roca, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+molecular+assay+of+tumor+radiosensitivity%3A+a+roadmap+towards+biology-based+personalized+radiation+therapy%5BTitle%5D)+AND+%22Per.+Med%22%5BJournal%5D)">A molecular assay of tumor radiosensitivity: a roadmap towards biology-based personalized radiation therapy.</a> Per. Med. 9, 547-557 (2012).</li>
</ol>

The authors declare that they have no competing financial interests.

The critical steps within the protocol are as follows. First, cells should be grown on the culture dish as a monolayer because conducting a morphological assessment of DAPI-stained nuclei is difficult for multilayered cells. To this end, in step 2.9, careful transfer of the culture dish to an incubator is recommended; shaking of the culture dish generates a swirl of suspended cells that leads to the concentration of cells at the center of the culture dish. In addition, overconfluence leading to multilayered cells should be avoided. To this end, in step 2.7, the number of cells seeded on the culture dish can be modified based on the population doubling time and the interval between irradiation and fixation. A confluence of approximately 80 at the time of fixation is recommended. Second, speed is important in fixation and DAPI staining (i.e., steps 4 and 5). Inter-sample inconsistency in regard to the time taken for these steps can lead to heterogeneity in DAPI signal intensity in the nuclei, which would obscure the morphological assessment.
In step 2.2, the number of cover slips in a single culture dish can be increased; a maximum of four cover slips can be placed in a 35 mm dish, and the number can be further increased using larger dishes. Placing multiple cover slips in each culture dish enables the efficient operation of time course assessment for a given treatment (i.e., the cover slips can be collected from a culture dish one by one at multiple time points of interest).
In step 3.2, the irradiation dose can be modified according to the researchers&#39; interest. The application of a consistent dose to multiple cell lines enables the comparison of the sensitivity to each mode of clonogenic cell death among the cell lines. On the other hand, the use of iso-clonogenic survival doses for each cell line enables comparison of clonogenic cell death profiles among the cell lines. The iso-clonogenic survival dose can be determined by the clonogenic survival assay12. The D10 value, the dose that provides 10% clonogenic survival, is a common endpoint for the iso-clonogenic survival dose.
In step 3.3, the time from irradiation to fixation can be modified according to the researchers&#39; interest; this is important because the peak time for IR-induced apoptosis, mitotic catastrophe, and cellular senescence varies according to cell line and treatment. In this article, we used 72 h after irradiation as the time point that would be most useful for the initial screening of clonogenic cell death profiles, based on multiple studies by our group and others described as follows1,2,3,4,8,9: (i) X-ray-induced apoptosis in cells established from solid tumors mostly occurs a few days after irradiation. (ii) X-ray-induced mitotic catastrophe in cancer cells occurs most prominently at the second or third mitosis after release from the temporary cell-cycle arrest induced by irradiation. The release usually occurs approximately 24 h after irradiation, followed by repeated mitoses at intervals of approximately 24 h. (iii) X-ray&#8211;induced cellular senescence becomes evident after an interval dependent upon the cell line in question: 2 days after irradiation for some early cases, and 7 days after irradiation for most cell lines. After obtaining an overall picture of the clonogenic cell death profiles from the initial screening, time course experiments will provide a more detailed elucidation of the peak time for each mode of clonogenic cell death in the specific cell line and/or condition of interest4.
It should be noted that the DAPI staining assay and the clonogenic survival assay, a gold standard method for radiation sensitivity assessment, are not interchangeable. Apoptosis and mitotic catastrophe only last for hours. Thus, the DAPI staining assay for a given time point detects apoptosis and mitotic catastrophe that occurs at the time point of the assessment. On the other hand, the results of the clonogenic survival assay at a given time point include the total amount of apoptosis and mitotic catastrophe that had occurred during the incubation period for typically 10 -&#160;14 days after irradiation. Different from apoptosis and mitotic catastrophe, senescent cells remain on the culture dish; they accumulate gradually over time after irradiation. Therefore, the results of both the DAPI staining assay and the clonogenic survival assay reflect the total amount of senescence that occurred during the incubation period. Importantly, the proportion of apoptosis, mitotic catastrophe, and senescence induced by irradiation varies widely according to cell line and irradiation dose. Taken together, theoretically, the results of one assay cannot be translated directly into those of the other assay.
The DAPI staining assay has a few limitations. First, it remains controversial whether mitotic catastrophe is a distinct mode of cell death. In the field of radiation biology, mitotic catastrophe is considered a major mode of IR-induced cell death that is distinct from other mechanisms of clonogenic cell death1. On the other hand, others argue that mitotic catastrophe is not a distinct mode of cell death but rather a process that precedes cell death including apoptosis and necrosis13,14. Thus, apoptosis and mitotic catastrophe may overlap to some extent. Second, previous studies suggest that cellular senescence can occur in the absence of SAHF in some cell lines and treatment settings2. At present, other assays specifically designed for each clonogenic cell death mode should be used to increase the robustness of the conclusions of a given experiment. Third, the DAPI staining assay cannot assess modes of clonogenic cell death other than apoptosis, mitotic catastrophe, and cellular senescence (e.g., necrosis and autophagy). Fourth, the utility of the DAPI staining assay as a predictor of tumor response to radiotherapy has not been elucidated in the clinic. From this point of view, the clonogenic survival assay, which assesses the total amount of clonogenic cell death, is superior to the DAPI staining assay because a correlation has been established between SF2, the surviving fraction of cells irradiated with 2 Gy X-rays, and the tumor response to radiotherapy15. Nevertheless, it is noteworthy that the clonogenic survival assay is not utilized widely in the clinic, mainly due to the requirement for a high degree of expertise and a long period of time (i.e., 14 days) for data acquisition. By comparison, the procedure for the DAPI staining assay is simpler and takes significantly less time, approximately 3 - 4 days, to generate results. The utility of the DAPI staining assay as a predictor of tumor response to radiotherapy will be tested in the clinic in the near future.
In summary, the DAPI staining assay is a cost-effective one-step assay to simultaneously assess the three major modes of IR-induced clonogenic cell death. This approach allows one to easily screen for the modes of clonogenic cell death for various cell lines, treatment settings, and time points, with the goal of elucidating the mechanisms of cell death in the target cells and conditions of interest.

Ionizing radiation (IR) induces multiple modes of clonogenic cell death. Research on IR-induced clonogenic cell death is important for understanding the toxicity of IR to normal tissues, as well as for developing methods to increase the treatment efficacy of cancer radiotherapy. Apoptosis, mitotic catastrophe, and cellular senescence are the major modes of clonogenic cell death induced by IR1. Apoptosis is a regulated mode of cell death that is initiated by DNA damage1. Mitotic catastrophe is cell death that occurs due to aberrant mitosis resulting from unrepaired DNA double-strand breaks1. Cellular senescence is defined as a state of irreversible cell growth arrest2; note that cellular senescence is not cell death per se, but it is a mode of clonogenic cell death because it abolishes the clonogenic survival of the senescent cell.
Various cell-based assays have been developed to individually assess apoptosis, mitotic catastrophe, and cellular senescence. Apoptosis can be assessed by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining, annexin V staining, DNA fragmentation assays, and sub-G1 cell-cycle phase determination by flow cytometry1. Mitotic catastrophe can be assessed by immunofluorescence staining for mitotic markers, including MPM2, TUNEL staining, and electron microscopy1. Cellular senescence can be assessed by senescence-associated &#946;-galactosidase (SA-&#946;-Gal) staining, growth-arrest assays, and electron microscopy1. Importantly, the predominant mode of clonogenic cell death differs among cell lines and treatment settings3,4. Therefore, to elucidate the overall profile of clonogenic cell death for a given experimental setting, multiple assays covering all of these modes of death must be conducted together, which is labor- and cost-intensive.
In this article, we describe a quick and cost-effective one-step assay for simultaneously assessing apoptosis, mitotic catastrophe, and cellular senescence induced by IR3,4. In this method, cells grown on a cover slip are irradiated with X-rays and stained with 4&#39;,6-diamidino-2-phenylindole dihydrochloride (DAPI). Using fluorescence microscopy, apoptosis, mitotic catastrophe, and cellular senescence are each identified based on the respective characteristic morphologies of the DAPI-stained nuclei. This approach will be useful for applications including screening for clonogenic cell death profiles in various cell lines, treatment settings, and time points, with the goal of investigating the mechanisms of cell death in the target cells and conditions of interest.

<table><tbody><tr><td>cover slip</td><td>Matsunami</td><td>C013001</td><td></td></tr><tr><td>paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148-1KG</td><td></td></tr><tr><td>sucrose</td><td>Sigma-Aldrich</td><td>84097-250G</td><td></td></tr><tr><td>phosphate-buffered saline</td><td>Cosmo Bio</td><td>16232000</td><td></td></tr><tr><td>scalpel</td><td>Kai Medical</td><td>No.20, 520-A</td><td></td></tr><tr><td>35 mm cell culture dish</td><td>Falcon</td><td>353001</td><td></td></tr><tr><td>trypsin</td><td>Wako</td><td>201-16945</td><td></td></tr><tr><td>inverted phase microscope</td><td>Shimazu</td><td>114-909</td><td></td></tr><tr><td>X-ray irradiator</td><td>Faxitron Bioptics</td><td>Faxitron RX-650</td><td></td></tr><tr><td>square culture dish</td><td>Corning</td><td>431110</td><td></td></tr><tr><td>slide glass</td><td>Matsunami</td><td>Pro-11</td><td></td></tr><tr><td>DAPI staining reagent</td><td>Cell Signaling</td><td>8961S</td><td></td></tr><tr><td>fluorescence microscope</td><td>Nikon</td><td>ECLIPSE Ni</td><td></td></tr><tr><td>fluorescence microscope DAPI filter</td><td>Nikon</td><td>U-FUW, BP340-390, BA420IF, DM410</td><td></td></tr><tr><td>fluorescence microscope lens (20&amp;times;)</td><td>Nikon</td><td>Plan Apo &amp;lambda; 20X/0.75</td><td></td></tr><tr><td>fluorescence microscope lens (60&amp;times;, oil)</td><td>Nikon</td><td>Plan Apo &amp;lambda; 60X/1.40 oil</td><td></td></tr><tr><td>oil</td><td>Olympus</td><td>N5218600</td><td></td></tr><tr><td>CCD camera</td><td>Nikon</td><td>DS-Qi2</td><td></td></tr><tr><td>digital image acquisition software</td><td>Nikon</td><td>NIS-Elements Document</td><td></td></tr><tr><td>lens cleaner</td><td>Olympus</td><td>OT0552</td><td></td></tr><tr><td>chloroform</td><td>Wako</td><td>035-02616</td><td></td></tr></tbody></table>

one-step protocol for evaluation of the mode of radiation-induced clonogenic cell death by fluorescence microscopy

Research on ionizing radiation (IR)-induced clonogenic cell death is important for understanding the effect of IR on malignant tumors and normal tissues. Here, we describe a quick and cost-effective one-step assay for simultaneously assessing the major modes of clonogenic cell death induced by IR, i.e., apoptosis, mitotic catastrophe, and cellular senescence. In this method, cells grown on a cover slip are irradiated with X-rays and stained with 4&#39;,6-diamidino-2-phenylindole dihydrochloride (DAPI). Using fluorescence microscopy, apoptosis, mitotic catastrophe, and cellular senescence are identified based on the characteristic morphologies of the DAPI-stained nuclei. Apoptosis is determined by the presence of apoptotic bodies (i.e., condensed and fragmented nuclei). Mitotic catastrophe is determined by the presence of nuclei that exhibit two or more distinct lobes and micronuclei. Cellular senescence is determined by the presence of senescence-associated heterochromatic foci (i.e., nuclear DNA containing 30-50 bright, dense foci). This approach allows the experimenter to easily screen for clonogenic cell death modes using various cell lines, treatment settings, and/or time points, with the goal of elucidating the mechanisms of cell death in the target cells and conditions of interest.

As an example, U2OS human osteosarcoma cells were treated with 6 Gy X-rays, incubated for 72 h, and subjected to the DAPI staining assay according to the protocol. Figure 1 shows zoomed images for the typical nuclear morphologies associated with apoptosis, mitotic catastrophe, and cellular senescence obtained using a 60&#215; oil lens. Refer to steps 7.1.1-7.1.3 for the characteristic morphologies for each mode of clonogenic cell death. Figure 2 shows overview images of nuclei obtained using a 20X&#160;lens. Irradiation with 6 Gy X-rays induced mitotic catastrophe frequently, apoptosis less frequently, and cellular senescence rarely. In this manner, examination at a low-magnification field allows one to apprehend at a glance the overall picture of the clonogenic cell death profile in a given experimental setting. Figure 3 shows a graphical representation of the quantified data.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56338/56338fig1.jpg" /> 
Figure 1: Zoomed images of DAPI-stained nuclei showing typical morphologies of apoptosis, mitotic catastrophe, and cellular senescence. U2OS cells were irradiated with 6 Gy X-rays. After 72 h, the cells were fixed and stained with DAPI. Nuclear images were acquired using a 60X oil lens. Scale bar = 20 &#956;m. <a href="https://cloudflare.jove.com/files/ftp_upload/56338/56338fig1large.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/56338/56338fig2.jpg" /> 
Figure 2: Overview images of DAPI-stained nuclei of cells treated with X-rays. U2OS cells were irradiated with 6 Gy X-rays or mock-irradiated. After 72 h, the cells were fixed and stained with DAPI. Images of nuclei were acquired using a 20X lens. Circles, apoptosis; arrows, mitotic catastrophe. Scale bar = 50 &#956;m. <a href="https://cloudflare.jove.com/files/ftp_upload/56338/56338fig2large.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/56338/56338fig3.jpg" /> 
Figure 3: Quantified data for apoptosis, mitotic catastrophe, and cellular senescence induced in cells treated with X-rays. U2OS cells were irradiated with 6 Gy X-rays or mock-irradiated. After 72 h, the cells were fixed and stained with DAPI. Images of nuclei were acquired using a 20X lens. From random fields, a total of 300 nuclei were evaluated for apoptosis (Ap), mitotic catastrophe (MC), and cellular senescence (Sns). The evaluations were performed in triplicate. Average values are shown, and error bars indicate standard deviations.&#160;<a href="https://cloudflare.jove.com/files/ftp_upload/56338/56338fig3large.jpg" style="font-size: 14px;" target="_blank">Please click here to view a larger version of this figure.</a>

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One-step Protocol for Evaluation of the Mode of Radiation-induced Clonogenic Cell Death by Fluorescence Microscopy

Research on ionizing radiation (IR)-induced clonogenic cell death is important for understanding the effects of IR on malignant tumors and normal tissues. Here, we describe a one-step assay for assessing the major modes of IR-induced clonogenic cell death based on morphology of 4&#39;,6-diamidino-2-phenylindole dihydrochloride (DAPI)-stained nuclei visualized by fluorescence microscopy.

Research on ionizing radiation (IR)-induced clonogenic cell death is important for understanding the effect of IR on malignant tumors and normal tissues. ...

one-step-protocol-for-evaluation-mode-radiation-induced-clonogenic

Research

JoVE Journal

Cancer Research

11.3K Views.  Gunma University Graduate School of Medicine. Research on ionizing radiation (IR)-induced clonogenic cell death is important for understanding the effects of IR on malignant tumors and normal tissues. Here, we describe a one-step assay for assessing the major modes of IR-induced clonogenic cell death based on morphology of 4',6-diamidino-2-phenylindole dihydrochloride (DAPI)-stained nuclei visualized by fluorescence microscopy.

Video: One-step Protocol for Evaluation of the Mode of Radiation-induced Clonogenic Cell Death by Fluorescence Microscopy

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

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation became a valuable experimental tool to investigate the DNA damage response (DDR) in vivo. It allows real-time high-resolution single-cell analysis of macromolecular dynamics in response to laser-induced damage confined to a submicrometer region in the cell nucleus. However, various laser conditions have been used without appreciation of differences in the types of damage induced. As a result, the nature of the damage is often not well characterized or controlled, causing apparent inconsistencies in the recruitment or modification profiles. We demonstrated that different irradiation conditions (i.e., different wavelengths as well as different input powers (irradiances) of a femtosecond (fs) near-infrared (NIR) laser) induced distinct DDR and repair protein assemblies. This reflects the type of DNA damage produced. This protocol describes how titration of laser input power allows induction of different amounts and complexities of DNA damage, which can easily be monitored by detection of base and crosslinking damages, differential poly (ADP-ribose) (PAR) signaling, and pathway-specific repair factor assemblies at damage sites. Once the damage conditions are determined, it is possible to investigate the effects of different damage complexity and differential damage signaling as well as depletion of upstream factor(s) on any factor of interest.

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

The goal of this protocol is to describe how to use laser microirradiation to induce different types of DNA damage, including relatively simple strand breaks and complex damage, to study DNA damage signaling and repair factor assembly at damage sites in vivo.

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation ...

Genetics

Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over the past decade, new and innovative technologies have greatly enhanced the practicality of live-cell imaging. Cells can now be kept in focus and continuously imaged over several days while maintained under 37 &#176;C and 5% CO2 cell culture conditions. Moreover, multiple fields of view representing different experimental conditions can be acquired simultaneously, thus providing high-throughput experimental data. Live-cell imaging provides a significant advantage over fixed-cell imaging by allowing for the direct visualization and temporal quantitation of dynamic cellular events. Live-cell imaging can also identify variation in the behavior of single cells that would otherwise have been missed using population-based assays. Here, we describe live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel. We demonstrate methods to visualize whether mitotically arrested cells die directly from mitosis or slip back into interphase. We also describe how the fluorescent ubiquitination-based cell cycle indicator (FUCCI) system can be used to assess the fraction of interphase cells born from mitotic slippage that are capable of re-entering the cell cycle. Finally, we describe a live-cell imaging method to identify nuclear envelope rupture events.

Live-cell imaging provides a wealth of information on single cells or whole populations that is unattainable by fixed cell imaging alone. Here, live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel are described.

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over ...

Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, senescence is a fundamentally different machinery restraining&#160;propagation of cancer cells. Decades of scientific studies have revealed the complex pathological effects of senescent cancer cells in tumors and microenvironments that modulate cancer cells and stromal cells. New evidence suggests that senescence is a potent prognostic factor during cancer treatment, and therefore rapid and accurate detection of senescent cells in cancer samples is essential. This paper presents a method to visualize and detect therapy-induced senescence (TIS) in cancer cells. Diffuse large B-cell lymphoma (DLBCL) cell lines were treated with mafosfamide (MAF) or daunorubicin (DN) and examined for the senescence marker, senescence-associated &#946;-galactosidase (SA-&#946;-gal), the DNA synthesis marker 5-ethynyl-2&#8242;-deoxyuridine (EdU), and the DNA damage marker gamma-H2AX (&#947;H2AX). Flow cytometer imaging can help generate high-resolution single-cell images in a short period of time to simultaneously visualize and quantify the three markers in cancer cells.

Here we present a flow cytometry-based method for visualization and quantification of multiple senescence-associated markers in single cells.

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, ...

Evaluating Quantitative Tumor Cell Killing by CAR-Expressing Jurkat Cells

Rapid In Vitro Cytotoxicity Evaluation of Jurkat Expressing Chimeric Antigen Receptor using Fluorescent Imaging

Chimeric antigen receptor (CAR) T cells are at the forefront of oncology. A CAR is constructed of a targeting domain (usually a single chain variable fragment, scFv), with an accompanying intra-chain linker, followed by a hinge, transmembrane, and costimulatory domain. Modification of the intra-chain linker and hinge domain can have a significant effect on CAR-mediated killing. Considering the many different options for each part of a CAR construct, there are large numbers of permutations. Making CAR-T cells is a time-consuming and expensive process, and making and testing many constructs is a heavy time and material investment. This protocol describes a platform to rapidly evaluate hinge-optimized CAR constructs in Jurkat cells (CAR-J). Jurkat cells are an immortalized T cell line with high lentivirus uptake, allowing for efficient CAR transduction. Here, we present a platform to rapidly evaluate CAR-J using a fluorescent imager, followed by confirmation of cytolysis in PBMC-derived T cells.

Author Spotlight: High-Throughput Screening of CAR T-Cell Constructs for Enhanced Cytotoxicity and Immunologic Memory 

A protocol to evaluate quantitative tumor cell killing by Jurkat cells expressing chimeric antigen receptor (CAR) targeting single tumor antigen. This protocol can be used as a screening platform for rapid optimization of CAR hinge constructs prior to confirmation in peripheral blood-derived T cells.

Chimeric antigen receptor (CAR) T cells are at the forefront of oncology. A CAR is constructed of a targeting domain (usually a single chain variable ...

Radiation Response in Patient-Derived Tumor Organoids Using a Clonogenic Assay

Live Imaging to Quantify Cellular Radiosensitivity in Patient-Derived Tumor Organoids

Radiation therapy (RT) is one of the mainstays of modern clinical cancer management. However, not all cancer types are equally sensitive to irradiation, often (but not always) because of differences in the ability of malignant cells to repair oxidative DNA damage as elicited by ionizing rays. Clonogenic assays have been employed for decades to assess the sensitivity of cultured cancer cells to ionizing irradiation, largely because irradiated cancer cells often die in a delayed manner that is difficult to quantify with short-term flow cytometry- or microscopy-assisted techniques. Unfortunately, clonogenic assays cannot be employed as such for more complex tumor models, such as patient-derived tumor organoids (PDTOs). Indeed, irradiating established PDTOs may not necessarily abrogate their growth as multicellular units, unless their stem-like compartment is completely eradicated. Moreover, irradiating PDTO-derived single-cell suspensions may not properly recapitulate the sensitivity of malignant cells to RT in the context of established PDTOs. Here, we detail an adaptation of conventional clonogenic assays that involves exposure of established PDTOs to ionizing radiation, followed by single-cell dissociation, replating in suitable culture conditions and live imaging. Non-irradiated (control) PDTO-derived stem-like cells reform growing PDTOs with a PDTO-specific efficiency, which is negatively influenced by irradiation in a dose-dependent manner. In these conditions, PDTO-forming efficiency and growth rate can be quantified as a measure of radiosensitivity on time-lapse images collected until control PDTOs achieve a predefined space occupancy.

Author Spotlight: Radiotherapy and Clonogenic Assays for Advancing Cancer Research and Personalized Medicine

Here, we detail a straightforward live imaging approach for quantifying the sensitivity of patient-derived tumor organoids to ionizing radiation.

Radiation therapy (RT) is one of the mainstays of modern clinical cancer management. However, not all cancer types are equally sensitive to irradiation, ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Biology

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

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.

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.

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological ...

Quantifying Cell Death in Human Colonoids in Response to Cytotoxic Perturbagens

Quantification of Cytokine-Induced Cell Death in Human Colonic Organoids Using Live Fluorescence Microscopy

Intestinal epithelial cell (IEC) death is increased in patients with inflammatory bowel diseases (IBD) such as ulcerative colitis (UC) and Crohn's disease (CD). This can contribute to defects in intestinal barrier function, exacerbation of inflammation, and disease immunopathogenesis. Cytokines and death receptor ligands are partially responsible for this increase in IEC death. IBD-relevant cytokines, such as TNF-&#945; and IFN-&#947;, are cytotoxic to IECs both independently and in combination. This protocol describes a simple and practical assay to quantify cytokine-induced cytotoxicity in CD patient-derived colonic organoids using a fluorescent cell death dye (SYTOX Green Nucleic Acid Stain), live fluorescence microscopy, and open-source image analysis software. We also demonstrate how to use the Bliss independence mathematical model to calculate a coefficient of perturbagen interaction (CPI) based on organoid cytotoxicity. The CPI can be used to determine if interactions between cytokine combinations or other types of perturbagens are antagonistic, additive, or synergistic. This protocol can be implemented to investigate the cytotoxic activity of cytokines and other perturbagens using patient-derived colonic organoids.

Author Spotlight: Understanding Cytokine-Induced Cell Death in Intestinal Epithelial Cells Using Human Organoids

This protocol describes a simple and cost-effective method to investigate and quantify cell death in human colonic organoids in response to cytotoxic perturbagens such as cytokines. The approach employs a fluorescent cell death dye (SYTOX Green Nucleic Acid Stain), live fluorescence microscopy, and open-source image analysis software to quantify single-organoid responses to cytotoxic stimuli.

Intestinal epithelial cell (IEC) death is increased in patients with inflammatory bowel diseases (IBD) such as ulcerative colitis (UC) and Crohn's disease ...

Assessing DNA Damage Foci: To Identify Radiation-Induced DNA Damage Foci in Cancer Cell Line Using Immunofluorescence Staining

Source: Maliszewska-Olejniczak et al. <a href="https://www.jove.com/v/61399"> Immunofluorescence Imaging of DNA Damage and Repair Foci in Human Colon Cancer Cells.</a> J. Vis. Exp. (2020).
This video describes a protocol to detect radiation-induced DNA repair foci in the colon cancer cell lines using immunofluorescence staining. DNA repair proteins get activated at the DNA damage site; therefore, we can detect them using specific antibodies and visualize them under a fluorescence microscope.

Source: Maliszewska-Olejniczak et al.  Immunofluorescence Imaging of DNA Damage and Repair Foci in Human Colon Cancer Cells. J. Vis. Exp. (2020).
This ...

JoVE Encyclopedia of Experiments

Colorectal Cancer

Assays & techniques

Cell Death Screening Using DAPI Staining: A Method for Rapid Screening of IR Induced Clonogenic Cell Death in Cancerous Cell Lines

Source: Kobayashi, D. et al. <a href="https://www.jove.com/video/56338" target="_blank">One-step Protocol for Evaluation of the Mode of Radiation-induced Clonogenic Cell Death by Fluorescence Microscopy.</a> J. Vis. Exp. (2017)
This video demonstrates the methodology to screen for major cell death profiles in ionizing radiation (IR) induced cancer cell lines using DAPI staining assay. The DAPI-stained nuclei of target cells undergoing cell death exhibit distinct morphologies and can be easily identified by fluorescence microscopy.

Source: Kobayashi, D. et al. One-step Protocol for Evaluation of the Mode of Radiation-induced Clonogenic Cell Death by Fluorescence Microscopy. J. Vis. ...

One-step Protocol for Evaluation of the Mode of Radiation-induced Clonogenic Cell Death by Fluorescence Microscopy

Summary

Explore More Videos

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

Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel

Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells

Rapid In Vitro Cytotoxicity Evaluation of Jurkat Expressing Chimeric Antigen Receptor using Fluorescent Imaging

Live Imaging to Quantify Cellular Radiosensitivity in Patient-Derived Tumor Organoids

Clonogenic Assay: Adherent Cells

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

Quantification of Cytokine-Induced Cell Death in Human Colonic Organoids Using Live Fluorescence Microscopy

Assessing DNA Damage Foci: To Identify Radiation-Induced DNA Damage Foci in Cancer Cell Line Using Immunofluorescence Staining

Cell Death Screening Using DAPI Staining: A Method for Rapid Screening of IR Induced Clonogenic Cell Death in Cancerous Cell Lines