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An overview of apoptosis assays detecting DNA fragmentation

Abstract

Apoptosis has been recognized as a type of programmed cell death connected with characteristic morphological and biochemical changes in cells. This programmed cell death plays an important role in the genesis of a number of physiological and pathological processes. Thus, it can be very important to detect the signs of apoptosis in a study of cellular metabolism. The present paper provides an overview of methods often being used for detecting DNA fragmentation as one of the most specific findings in apoptosis. To date, three routine assays have been developed for detecting DNA fragmentation: DNA ladder assay, TUNEL assay, and comet assay. All these methods differ in their principles for detecting DNA fragmentation. DNA ladder assay detects the characteristic “DNA ladder” pattern formed during internucleosomal cleavage of DNA. Terminal deoxynUcleotidyl transferase Nick-End Labeling (TUNEL) assay detects DNA strand breaks using terminal deoxynucleotidyl transferase catalyzing attachment of modified deoxynucleotides on the DNA strand breaks. Comet assay can be used for detecting nucleus breakdown producing single/double-strand DNA breaks. The aim of this review is to describe the present knowledge on these three methods, including optimized approaches, techniques, and limitations.

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References

  1. 1.

    Kerr JF, Wyllie AH, Currie AR (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26(4):239–257

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  2. 2.

    Elmore S (2007) Apoptosis: a review of programmed cell death. Toxicol Pathol 35(4):495–516. https://doi.org/10.1080/01926230701320337

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  3. 3.

    Choudhary GS, Al-Harbi S, Almasan A (2015) Caspase-3 activation is a critical determinant of genotoxic stress-induced apoptosis. Methods Mol Biol 1219:1–9. https://doi.org/10.1007/978-1-4939-1661-0_1

    CAS  PubMed  Article  Google Scholar 

  4. 4.

    McIlwain DR, Berger T, Mak TW (2013) Caspase functions in cell death and disease. Cold Spring Harb Perspect Biol 5(4):a008656. https://doi.org/10.1101/cshperspect.a008656

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  5. 5.

    Li J, Yuan J (2008) Caspases in apoptosis and beyond. Oncogene 27(48):6194–6206. https://doi.org/10.1038/onc.2008.297

    CAS  PubMed  Article  Google Scholar 

  6. 6.

    Mattson MP (2000) Apoptosis in neurodegenerative disorders. Nat Rev Mol Cell Bio 1(2):120–129. https://doi.org/10.1038/35040009

    CAS  Article  Google Scholar 

  7. 7.

    Wong RSY (2011) Apoptosis in cancer: from pathogenesis to treatment. J Exp Clin Canc Res 30. https://doi.org/10.1186/1756-9966-30-87

  8. 8.

    Saraste A, Pulkki K (2000) Morphologic and biochemical hallmarks of apoptosis. Cardiovasc Res 45(3):528–537

    CAS  Article  Google Scholar 

  9. 9.

    Boe R, Gjertsen BT, Vintermyr OK, Houge G, Lanotte M, Doskeland SO (1991) The protein phosphatase inhibitor okadaic acid induces morphological changes typical of apoptosis in mammalian cells. Exp Cell Res 195(1):237–246

    CAS  PubMed  Article  Google Scholar 

  10. 10.

    Birkinshaw RW, Czabotar PE (2017) The BCL-2 family of proteins and mitochondrial outer membrane permeabilisation. Semin Cell Dev Biol 72:152–162. https://doi.org/10.1016/j.semcdb.2017.04.001

    CAS  PubMed  Article  Google Scholar 

  11. 11.

    Hacker G (2000) The morphology of apoptosis. Cell Tissue Res 301(1):5–17

    CAS  PubMed  Article  Google Scholar 

  12. 12.

    Takano YS, Harmon BV, Kerr JFR (1991) Apoptosis induced by mild hyperthermia in human and murine tumor-cell lines—a study using electron-microscopy and DNA gel-electrophoresis. J Pathol 163(4):329–336. https://doi.org/10.1002/path.1711630410

    CAS  PubMed  Article  Google Scholar 

  13. 13.

    Wyllie AH (1980) Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284(5756):555–556

    CAS  PubMed  Article  Google Scholar 

  14. 14.

    Yasuhara S, Zhu Y, Matsui T, Tipirneni N, Yasuhara Y, Kaneki M, Rosenzweig A, Martyn JA (2003) Comparison of comet assay, electron microscopy, and flow cytometry for detection of apoptosis. J Histochem Cytochem 51(7):873–885. https://doi.org/10.1177/002215540305100703

    CAS  PubMed  Article  Google Scholar 

  15. 15.

    Rahman Q, Lohani M, Dopp E, Pemsel H, Jonas L, Weiss DG, Schiffmann D (2002) Evidence that ultrafine titanium dioxide induces micronuclei and apoptosis in Syrian hamster embryo fibroblasts. Environ Health Perspect 110(8):797–800. doi:sc271_5_1835

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  16. 16.

    Burattini S, Falcieri E (2013) Analysis of cell death by electron microscopy. Methods Mol Biol 1004:77–89. https://doi.org/10.1007/978-1-62703-383-1_7

    CAS  PubMed  Article  Google Scholar 

  17. 17.

    Pesce M, De Felici M (1994) Apoptosis in mouse primordial germ cells: a study by transmission and scanning electron microscope. Anat Embryol (Berl) 189(5):435–440

    CAS  Article  Google Scholar 

  18. 18.

    Loo DT, Copani A, Pike CJ, Whittemore ER, Walencewicz AJ, Cotman CW (1993) Apoptosis is induced by beta-amyloid in cultured central-nervous-system neurons. Proc Natl Acad Sci USA 90(17):7951–7955. https://doi.org/10.1073/pnas.90.17.7951

    CAS  PubMed  Article  Google Scholar 

  19. 19.

    Hessler JA, Budor A, Putchakayala K, Mecke A, Rieger D, Banaszak Holl MM, Orr BG, Bielinska A, Beals J, Baker J Jr (2005) Atomic force microscopy study of early morphological changes during apoptosis. Langmuir 21(20):9280–9286. https://doi.org/10.1021/la051837g

    CAS  PubMed  Article  Google Scholar 

  20. 20.

    Kuznetsov YG, Malkin AJ, McPherson A (1997) Atomic force microscopy studies of living cells: visualization of motility, division, aggregation, transformation, and apoptosis. J Struct Biol 120(2):180–191. https://doi.org/10.1006/jsbi.1997.3936

    CAS  PubMed  Article  Google Scholar 

  21. 21.

    Henry CM, Hollville E, Martin SJ (2013) Measuring apoptosis by microscopy and flow cytometry. Methods 61(2):90–97. https://doi.org/10.1016/j.ymeth.2013.01.008

    CAS  PubMed  Article  Google Scholar 

  22. 22.

    Fadok VA, Bratton DL, Frasch SC, Warner ML, Henson PM (1998) The role of phosphatidylserine in recognition of apoptotic cells by phagocytes. Cell Death Differ 5(7):551–562. https://doi.org/10.1038/sj.cdd.4400404

    CAS  PubMed  Article  Google Scholar 

  23. 23.

    Baskic D, Popovic S, Ristic P, Arsenijevic NN (2006) Analysis of cycloheximide-induced apoptosis in human leukocytes: fluorescence microscopy using annexin V/propidium iodide versus acridin orange/ethidium bromide. Cell Biol Int 30(11):924–932. https://doi.org/10.1016/j.cellbi.2006.06.016

    CAS  PubMed  Article  Google Scholar 

  24. 24.

    Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C (1995) A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J Immunol Methods 184(1):39–51

    CAS  PubMed  Article  Google Scholar 

  25. 25.

    Sawai H, Domae N (2011) Discrimination between primary necrosis and apoptosis by necrostatin-1 in Annexin V-positive/propidium iodide-negative cells. Biochem Biophys Res Commun 411(3):569–573. https://doi.org/10.1016/j.bbrc.2011.06.186

    CAS  PubMed  Article  Google Scholar 

  26. 26.

    Eriksson S, Kim SK, Kubista M, Norden B (1993) Binding of 4′,6-diamidino-2-phenylindole (DAPI) to AT regions of DNA: evidence for an allosteric conformational change. Biochemistry 32(12):2987–2998

    CAS  PubMed  Article  Google Scholar 

  27. 27.

    Martin RM, Leonhardt H, Cardoso MC (2005) DNA labeling in living cells. Cytometry Part A 67(1):45–52. https://doi.org/10.1002/cyto.a.20172

    CAS  Article  Google Scholar 

  28. 28.

    Kajstura M, Halicka HD, Pryjma J, Darzynkiewicz Z (2007) Discontinuous fragmentation of nuclear DNA during apoptosis revealed by discrete “sub-G(1)” peaks on DNA content histograms. Cytometry Part A 71A(3):125–131. https://doi.org/10.1002/cyto.a.20357

    CAS  Article  Google Scholar 

  29. 29.

    Yoshida T, Konishi M, Horinaka M, Yasuda T, Goda AE, Taniguchi H, Yano K, Wakada M, Sakai T (2008) Kaempferol sensitizes colon cancer cells to TRAIL-induced apoptosis. Biochem Biophys Res Commun 375(1):129–133. https://doi.org/10.1016/j.bbrc.2008.07.131

    CAS  PubMed  Article  Google Scholar 

  30. 30.

    Chang CY, Li JR, Wu CC, Wang JD, Yang CP, Chen WY, Wang WY, Chen CJ (2018) Indomethacin induced glioma apoptosis involving ceramide signals. Exp Cell Res. https://doi.org/10.1016/j.yexcr.2018.02.019

    PubMed  Article  Google Scholar 

  31. 31.

    Liu XS, Zou H, Slaughter C, Wang XD (1997) DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis. Cell 89(2):175–184. https://doi.org/10.1016/S0092-8674(00)80197-X

    CAS  PubMed  Article  Google Scholar 

  32. 32.

    Widlak P (2000) The DFF40/CAD endonuclease and its role in apoptosis. Acta Biochim Pol 47(4):1037–1044

    CAS  PubMed  Google Scholar 

  33. 33.

    Widlak P, Li P, Wang X, Garrard WT (2000) Cleavage preferences of the apoptotic endonuclease DFF40 (caspase-activated DNase or nuclease) on naked DNA and chromatin substrates. J Biol Chem 275(11):8226–8232

    CAS  PubMed  Article  Google Scholar 

  34. 34.

    Nagata S, Nagase H, Kawane K, Mukae N, Fukuyama H (2003) Degradation of chromosomal DNA during apoptosis. Cell Death Differ 10(1):108–116. https://doi.org/10.1038/sj.cdd.4401161

    CAS  Article  Google Scholar 

  35. 35.

    Nagata S (2000) Apoptotic DNA fragmentation. Exp Cell Res 256(1):12–18. https://doi.org/10.1006/excr.2000.4834

    CAS  Article  Google Scholar 

  36. 36.

    Enari M, Sakahira H, Yokoyama H, Okawa K, Iwamatsu A, Nagata S (1998) A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD (vol 391, pg 43, 1998). Nature 393(6683):396–396. https://doi.org/10.1038/30782

    CAS  Article  Google Scholar 

  37. 37.

    Skalka M, Matyasova J, Cejkova M (1976) DNA in chromatin of irradiated lymphoid-tissues degrades invivo into regular fragments. FEBS Lett 72(2):271–274. https://doi.org/10.1016/0014-5793(76)80984-2

    CAS  PubMed  Article  Google Scholar 

  38. 38.

    Saadat YR, Saeidi N, Vahed SZ, Barzegari A, Barar J (2015) An update to DNA ladder assay for apoptosis detection. Bioimpacts 5(1):25–28

    CAS  Google Scholar 

  39. 39.

    Herrmann M, Lorenz HM, Voll R, Grunke M, Woith W, Kalden JR (1994) A rapid and simple method for the isolation of apoptotic DNA fragments. Nucleic Acids Res 22(24):5506–5507

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  40. 40.

    Yawata A, Adachi M, Okuda H, Naishiro Y, Takamura T, Hareyama M, Takayama S, Reed JC, Imai K (1998) Prolonged cell survival enhances peritoneal dissemination of gastric cancer cells. Oncogene 16(20):2681–2686. https://doi.org/10.1038/sj.onc.1201792

    CAS  PubMed  Article  Google Scholar 

  41. 41.

    Samarghandian S, Shabestari MM (2013) DNA fragmentation and apoptosis induced by safranal in human prostate cancer cell line. Indian J Urol 29(3):177–183. https://doi.org/10.4103/0970-1591.117278

    PubMed  PubMed Central  Article  Google Scholar 

  42. 42.

    Suman S, Pandey A, Chandna S (2012) An improved non-enzymatic “DNA ladder assay” for more sensitive and early detection of apoptosis. Cytotechnology 64(1):9–14. https://doi.org/10.1007/s10616-011-9395-0

    PubMed  Article  Google Scholar 

  43. 43.

    Yang TM, Qi SN, Zhao N, Yang YJ, Yuan HQ, Zhang B, Jin S (2013) Induction of apoptosis through caspase-independent or caspase-9-dependent pathway in mouse and human osteosarcoma cells by a new nitroxyl spin-labeled derivative of podophyllotoxin. Apoptosis 18(6):727–738. https://doi.org/10.1007/s10495-013-0819-5

    CAS  PubMed  Article  Google Scholar 

  44. 44.

    Takaki K, Higuchi Y, Hashii M, Ogino C, Shimizu N (2014) Induction of apoptosis associated with chromosomal DNA fragmentation and caspase-3 activation in leukemia L1210 cells by TiO2 nanoparticles. J Biosci Bioeng 117(1):129–133. https://doi.org/10.1016/j.jbiosc.2013.06.003

    CAS  PubMed  Article  Google Scholar 

  45. 45.

    Patel N, Joseph C, Corcoran GB, Ray SD (2010) Silymarin modulates doxorubicin-induced oxidative stress, Bcl-xL and p53 expression while preventing apoptotic and necrotic cell death in the liver. Toxicol Appl Pharmacol 245(2):143–152. https://doi.org/10.1016/j.taap.2010.02.002

    CAS  PubMed  Article  Google Scholar 

  46. 46.

    Micoud F, Mandrand B, Malcus-Vocanson C (2001) Comparison of several techniques for the detection of apoptotic astrocytes in vitro. Cell Proliferat 34(2):99–113

    CAS  Article  Google Scholar 

  47. 47.

    Cimmino A, Calin GA, Fabbri M, Iorio MV, Ferracin M, Shimizu M, Wojcik SE, Aqeilan RI, Zupo S, Dono M, Rassenti L, Alder H, Volinia S, Liu CG, Kipps TJ, Negrini M, Croce CM (2005) miR-15 and miR-16 induce apoptosis by targeting BCL2. Proc Natl Acad Sci USA 102(39):13944–13949. https://doi.org/10.1073/pnas.0506654102

    CAS  PubMed  Article  Google Scholar 

  48. 48.

    Kasahara Y, Tuder RM, Taraseviciene-Stewart L, Le Cras TD, Abman S, Hirth PK, Waltenberger J, Voelkel NF (2000) Inhibition of VEGF receptors causes lung cell apoptosis and emphysema. J Clin Invest 106(11):1311–1319. https://doi.org/10.1172/JCI10259

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  49. 49.

    Wang L, Sloper DT, Addo SN, Tian D, Slaton JW, Xing C (2008) WL-276, an antagonist against Bcl-2 proteins, overcomes drug resistance and suppresses prostate tumor growth. Cancer Res 68(11):4377–4383. https://doi.org/10.1158/0008-5472.CAN-07-6590

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  50. 50.

    Smina TP, Nitha B, Devasagayam TPA, Janardhanan KK (2017) Ganoderma lucidum total triterpenes induce apoptosis in MCF-7 cells and attenuate DMBA induced mammary and skin carcinomas in experimental animals. Mutat Res Genet Toxicol Environ Mutat 813:45–51. https://doi.org/10.1016/j.mrgentox.2016.11.010

    CAS  Article  Google Scholar 

  51. 51.

    Ahmad J, Alhadlaq HA, Siddiqui MA, Saquib Q, Al-Khedhairy AA, Musarrat J, Ahamed M (2015) Concentration-dependent induction of reactive oxygen species, cell cycle arrest and apoptosis in human liver cells after nickel nanoparticles exposure. Environ Toxicol 30(2):137–148. https://doi.org/10.1002/tox.21879

    CAS  PubMed  Article  Google Scholar 

  52. 52.

    Sunatani Y, Kamdar RP, Sharma MK, Matsui T, Sakasai R, Hashimoto M, Ishigaki Y, Matsumoto Y, Iwabuchi K (2018) Caspase-mediated cleavage of X-ray repair cross-complementing group 4 promotes apoptosis by enhancing nuclear translocation of caspase-activated DNase. Exp Cell Res 362(2):450–460. https://doi.org/10.1016/j.yexcr.2017.12.009

    CAS  PubMed  Article  Google Scholar 

  53. 53.

    Gavrieli Y, Sherman Y, Ben-Sasson SA (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119(3):493–501

    CAS  PubMed  Article  Google Scholar 

  54. 54.

    Gottschalk S, Anderson N, Hainz C, Eckhardt SG, Serkova NJ (2004) Imatinib (STI571)-mediated changes in glucose metabolism in human leukemia BCR-ABL-positive cells. Clin Cancer Res 10(19):6661–6668. https://doi.org/10.1158/1078-0432.Ccr-04-0039

    CAS  PubMed  Article  Google Scholar 

  55. 55.

    Pariente R, Pariente JA, Rodriguez AB, Espino J (2016) Melatonin sensitizes human cervical cancer HeLa cells to cisplatin-induced cytotoxicity and apoptosis: effects on oxidative stress and DNA fragmentation. J Pineal Res 60(1):55–64. https://doi.org/10.1111/jpi.12288

    CAS  PubMed  Article  Google Scholar 

  56. 56.

    Kim T, Jung U, Cho DY, Chung AS (2001) Se-methylselenocysteine induces apoptosis through caspase activation in HL-60 cells. Carcinogenesis 22(4):559–565

    CAS  PubMed  Article  Google Scholar 

  57. 57.

    Gorczyca W, Bruno S, Darzynkiewicz RJ, Gong JP, Darzynkiewicz Z (1992) DNA strand breaks occurring during apoptosis—their early insitu detection by the terminal deoxynucleotidyl transferase and nick translation assays and prevention by serine protease inhibitors. Int J Oncol 1(6):639–648

    CAS  PubMed  Google Scholar 

  58. 58.

    Gold R, Schmied M, Rothe G, Zischler H, Breitschopf H, Wekerle H, Lassmann H (1993) Detection of DNA fragmentation in apoptosis: application of in situ nick translation to cell culture systems and tissue sections. J Histochem Cytochem 41(7):1023–1030. https://doi.org/10.1177/41.7.8515045

    CAS  PubMed  Article  Google Scholar 

  59. 59.

    Li X, Darzynkiewicz Z (1995) Labelling DNA strand breaks with BrdUTP. Detection of apoptosis and cell proliferation. Cell Proliferat 28(11):571–579

    CAS  Article  Google Scholar 

  60. 60.

    Janicke RU, Sprengart ML, Wati MR, Porter AG (1998) Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis. J Biol Chem 273(16):9357–9360. https://doi.org/10.1074/jbc.273.16.9357

    CAS  PubMed  Article  Google Scholar 

  61. 61.

    Rengarajan T, Nandakumar N, Rajendran P, Haribabu L, Nishigaki I, Balasubramanian MP (2014) D-Pinitol promotes apoptosis in MCF-7 cells via induction of p53 and Bax and inhibition of Bcl-2 and NF-kappa B. Asian Pac J Cancer Prev 15(4):1757–1762. https://doi.org/10.7314/Apjcp.2014.15.4.1757

    PubMed  Article  Google Scholar 

  62. 62.

    Wang XQ, Wang L, Tan XR, Zhang HR, Sun GB (2014) Construction of doxorubicin-loading magnetic nanocarriers for assaying apoptosis of glioblastoma cells. J Colloid Interface Sci 436:267–275. https://doi.org/10.1016/j.jcis.2014.09.002

    CAS  PubMed  Article  Google Scholar 

  63. 63.

    Ajji PK, Binder MJ, Walder K, Puri M (2017) Balsamin induces apoptosis in breast cancer cells via DNA fragmentation and cell cycle arrest. Mol Cell Biochem 432(1–2):189–198. https://doi.org/10.1007/s11010-017-3009-x

    CAS  PubMed  Article  Google Scholar 

  64. 64.

    Gong JP, Traganos F, Darzynkiewicz Z (1994) A selective procedure for DNA extraction from apoptotic cells applicable for gel-electrophoresis and flow-cytometry. Anal Biochem 218(2):314–319. https://doi.org/10.1006/abio.1994.1184

    CAS  PubMed  Article  Google Scholar 

  65. 65.

    Ansari B, Coates PJ, Greenstein BD, Hall PA (1993) In situ end-labelling detects DNA strand breaks in apoptosis and other physiological and pathological states. J Pathol 170(1):1–8. https://doi.org/10.1002/path.1711700102

    CAS  PubMed  Article  Google Scholar 

  66. 66.

    Kok YJ, Swe M, Sit KH (2002) Necrosis has orderly DNA fragmentations. Biochem Biophys Res Commun 294(5):934–939. doi: S0006-291x(02)00587-9

    CAS  PubMed  Article  Google Scholar 

  67. 67.

    Mahassni SH, Al-Reemi RM (2013) Apoptosis and necrosis of human breast cancer cells by an aqueous extract of garden cress (Lepidium sativum) seeds. Saudi J Biol Sci 20(2):131–139. https://doi.org/10.1016/j.sjbs.2012.12.002

    PubMed  PubMed Central  Article  Google Scholar 

  68. 68.

    Collins AR, Ma AG, Duthie SJ (1995) The kinetics of repair of oxidative DNA-damage (strand breaks and oxidized pyrimidines) in human-cells. Mutat Res-DNA Repair 336(1):69–77. https://doi.org/10.1016/0921-8777(94)00043-6

    CAS  PubMed  Article  Google Scholar 

  69. 69.

    Cohen GM, Sun XM, Snowden RT, Dinsdale D, Skilleter DN (1992) Key morphological features of apoptosis May occur in the absence of internucleosomal DNA fragmentation. Biochem J 286:331–334. https://doi.org/10.1042/Bj2860331

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  70. 70.

    Collins AR (2004) The comet assay for DNA damage and repair: principles, applications, and limitations. Mol Biotechnol 26(3):249–261. https://doi.org/10.1385/MB:26:3:249

    CAS  PubMed  Article  Google Scholar 

  71. 71.

    Speit G, Hartmann A (1999) The comet assay (single-cell gel test). A sensitive genotoxicity test for the detection of DNA damage and repair. Methods Mol Biol 113:203–212. https://doi.org/10.1385/1-59259-675-4:203

    CAS  PubMed  Article  Google Scholar 

  72. 72.

    Rydberg B, Johnson JK (1978) Estimation of DNA strand breaks in single mammalian cells. Elsevier Inc., Amsterdam. https://doi.org/10.1016/B978-0-12-322650-1.50090-4

    Book  Google Scholar 

  73. 73.

    Ostling O, Johanson KJ (1984) Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem Biophys Res Commun 123(1):291–298

    CAS  PubMed  Article  Google Scholar 

  74. 74.

    Olive PL, Frazer G, Banath JP (1993) Radiation-induced apoptosis measured in TK6 human B lymphoblast cells using the comet assay. Radiat Res 136(1):130–136

    CAS  PubMed  Article  Google Scholar 

  75. 75.

    Olive PL, Banath JP (2006) The comet assay: a method to measure DNA damage in individual cells. Nat Protoc 1(1):23–29. https://doi.org/10.1038/nprot.2006.5

    CAS  PubMed  Article  Google Scholar 

  76. 76.

    Singh NP, McCoy MT, Tice RR, Schneider EL (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175(1):184–191

    CAS  PubMed  Article  Google Scholar 

  77. 77.

    Kim BM, Rode AB, Han EJ, Hong IS, Hong SH (2012) 5-Phenylselenyl- and 5-methylselenyl-methyl-2′-deoxyuridine induce oxidative stress, DNA damage, and caspase-2-dependent apoptosis in cancer cells. Apoptosis 17(2):200–216. https://doi.org/10.1007/s10495-011-0665-2

    CAS  PubMed  Article  Google Scholar 

  78. 78.

    Cortes-Gutierrez EI, Fernandez JL, Davila-Rodriguez MI, Lopez-Fernandez C, Gosalvez J (2017) Two-Tailed Comet Assay (2T-Comet): simultaneous detection of DNA single and double strand breaks. Methods Mol Biol 1560:285–293. https://doi.org/10.1007/978-1-4939-6788-9_22

    CAS  PubMed  Article  Google Scholar 

  79. 79.

    Rousset N, Keminon E, Eleouet S, Le Neel T, Auget JL, Vonarx V, Carre J, Lajat Y, Patrice T (2000) Use of alkaline Comet assay to assess DNA repair after m-THPC-PDT. J Photochem Photobiol B 56(2–3):118–131

    CAS  PubMed  Article  Google Scholar 

  80. 80.

    Mastaloudis A, Yu TW, O’Donnell RP, Frei B, Dashwood RH, Traber MG (2004) Endurance exercise results in DNA damage as detected by the comet assay. Free Radic Biol Med 36(8):966–975. https://doi.org/10.1016/j.freeradbiomed.2004.01.012

    CAS  PubMed  Article  Google Scholar 

  81. 81.

    Nandhakumar S, Parasuraman S, Shanmugam MM, Rao KR, Chand P, Bhat BV (2011) Evaluation of DNA damage using single-cell gel electrophoresis (Comet Assay). J Pharmacol Pharmacother 2(2):107–111. https://doi.org/10.4103/0976-500X.81903

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  82. 82.

    Chohan KR, Griffin JT, Lafromboise M, De Jonge CJ, Carrell DT (2006) Comparison of chromatin assays for DNA fragmentation evaluation in human sperm. J Androl 27(1):53–59. https://doi.org/10.2164/jandrol.05068

    CAS  PubMed  Article  Google Scholar 

  83. 83.

    Wilkins RC, Kutzner BC, Truong M, Sanchez-Dardon J, McLean JR (2002) Analysis of radiation-induced apoptosis in human lymphocytes: flow cytometry using Annexin V and propidium iodide versus the neutral comet assay. Cytometry 48(1):14–19. https://doi.org/10.1002/cyto.10098

    CAS  PubMed  Article  Google Scholar 

  84. 84.

    Schonn I, Hennesen J, Dartsch DC (2010) Cellular responses to etoposide: cell death despite cell cycle arrest and repair of DNA damage. Apoptosis 15(2):162–172. https://doi.org/10.1007/s10495-009-0440-9

    CAS  PubMed  Article  Google Scholar 

  85. 85.

    Hong YH, Jeon HL, Ko KY, Kim J, Yi JS, Ahn I, Kim TS, Lee JK (2018) Assessment of the predictive capacity of the optimized in vitro comet assay using HepG2 cells. Mutat Res 827:59–67. https://doi.org/10.1016/j.mrgentox.2018.01.010

    CAS  Article  Google Scholar 

  86. 86.

    Wang JL, Wang PC (2012) The effect of aging on the DNA damage and repair capacity in 2BS cells undergoing oxidative stress. Mol Biol Rep 39(1):233–241. https://doi.org/10.1007/s11033-011-0731-4

    CAS  PubMed  Article  Google Scholar 

  87. 87.

    Zhang H, Spitz MR, Tomlinson GE, Schabath MB, Minna JD, Wu X (2002) Modification of lung cancer susceptibility by green tea extract as measured by the comet assay. Cancer Detect Prev 26(6):411–418

    CAS  PubMed  Article  Google Scholar 

  88. 88.

    Konig K, Krasieva T, Bauer E, Fiedler U, Berns MW, Tromberg BJ, Greulich KO (1996) UVA-induced oxidative stress in single cells probed by autofluorescence modification, cloning assay, and comet assay. In: Proceedings of optical and imaging techniques for biomonitoring, vol 2628, pp 43–50

  89. 89.

    Kizilian N, Wilkins RC, Reinhardt P, Ferrarotto C, McLean JR, McNamee JP (1999) Silver-stained comet assay for detection of apoptosis. Biotechniques 27(5):926–930

    CAS  PubMed  Article  Google Scholar 

  90. 90.

    Moller P, Loft S, Ersson C, Koppen G, Dusinska M, Collins A (2014) On the search for an intelligible comet assay descriptor. Front Genet 5:217. https://doi.org/10.3389/fgene.2014.00217

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  91. 91.

    Fairbairn DW, Olive PL, O’Neill KL (1995) The comet assay: a comprehensive review. Mutat Res 339(1):37–59

    CAS  PubMed  Article  Google Scholar 

  92. 92.

    Konca K, Lankoff A, Banasik A, Lisowska H, Kuszewski T, Gozdz S, Koza Z, Wojcik A (2003) A cross-platform public domain PC image-analysis program for the comet assay. Mutat Res 534(1–2):15–20

    CAS  PubMed  Article  Google Scholar 

  93. 93.

    Barbisan LF, Scolastici C, Miyamoto M, Salvadori DM, Ribeiro LR, da Eira AF, de Camargo JL (2003) Effects of crude extracts of Agaricus blazei on DNA damage and on rat liver carcinogenesis induced by diethylnitrosamine. Genet Mol Res 2(3):295–308

    PubMed  Google Scholar 

  94. 94.

    Choucroun P, Gillet D, Dorange G, Sawicki B, Dewitte JD (2001) Comet assay and early apoptosis. Mutat Res 478(1–2):89–96

    CAS  PubMed  Article  Google Scholar 

  95. 95.

    Darzynkiewicz Z, Galkowski D, Zhao H (2008) Analysis of apoptosis by cytometry using TUNEL assay. Methods 44(3):250–254. https://doi.org/10.1016/j.ymeth.2007.11.008

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  96. 96.

    Lavolpe M, Lorenzi D, Greco E, Nodar F, Alvarez Sedo C (2015) Relationship between sperm DNA fragmentation and nuclear vacuoles. JBRA Assist Reprod 19(2):70–74. https://doi.org/10.5935/1518-0557.20150016

    PubMed  Article  Google Scholar 

  97. 97.

    Wang Y, Huang G, Wang Z, Qin H, Mo B, Wang C (2018) Elongation factor-2 kinase acts downstream of p38 MAPK to regulate proliferation, apoptosis and autophagy in human lung fibroblasts. Exp Cell Res. https://doi.org/10.1016/j.yexcr.2018.01.019

    PubMed  PubMed Central  Article  Google Scholar 

  98. 98.

    Ye X, Lin JY, Lin ZB, Xue AM, Li LL, Zhao ZQ, Liu L, Shen YW, Cong B (2017) Axin1 up-regulated 1 accelerates stress-induced cardiomyocytes apoptosis through activating Wnt/beta-catenin signaling. Exp Cell Res 359(2):441–448. https://doi.org/10.1016/l.yexcr.2017.08.027

    CAS  PubMed  Article  Google Scholar 

  99. 99.

    Grasl-Kraupp B, Ruttkay-Nedecky B, Koudelka H, Bukowska K, Bursch W, Schulte-Hermann R (1995) In situ detection of fragmented DNA (TUNEL assay) fails to discriminate among apoptosis, necrosis, and autolytic cell death: a cautionary note. Hepatology 21(5):1465–1468

    CAS  PubMed  Google Scholar 

  100. 100.

    Ribeiro S, Sharma R, Gupta S, Cakar Z, De Geyter C, Agarwal A (2017) Inter- and intra-laboratory standardization of TUNEL assay for assessment of sperm DNA fragmentation. Andrology 5(3):477–485. https://doi.org/10.1111/andr.12334

    CAS  PubMed  Article  Google Scholar 

  101. 101.

    Cuello-Carrion FD, Ciocca DR (1999) Improved detection of apoptotic cells using a modified in situ TUNEL technique. J Histochem Cytochem 47(6):837–839. https://doi.org/10.1177/002215549904700614

    CAS  PubMed  Article  Google Scholar 

  102. 102.

    Kuhn HG, DickinsonAnson H, Gage FH (1996) Neurogenesis in the dentate gyrus of the adult rat: age-related decrease of neuronal progenitor proliferation. J Neurosci 16(6):2027–2033

    CAS  PubMed  Article  Google Scholar 

  103. 103.

    Reagan-Shaw S, Ahmad N (2005) Silencing of polo-like kinase (Plk) 1 via siRNA causes induction of apoptosis and impairment of mitosis machinery in human prostate cancer cells: implications for the treatment of prostate cancer. FASEB J 19(6):611–613. https://doi.org/10.1096/fj.04-2910fje

    CAS  PubMed  Article  Google Scholar 

  104. 104.

    Darzynkiewicz Z, Zhao H (2011) Detection of DNA strand breaks in apoptotic cells by flow- and image-cytometry. Methods Mol Biol 682:91–101. https://doi.org/10.1007/978-1-60327-409-8_8

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  105. 105.

    Wu L, Prins HJ, Helder MN, van Blitterswijk CA, Karperien M (2012) Trophic effects of mesenchymal stem cells in chondrocyte co-cultures are independent of culture conditions and cell sources. Tissue Eng Part A 18(15–16):1542–1551. https://doi.org/10.1089/ten.tea.2011.0715

    CAS  PubMed  Article  Google Scholar 

  106. 106.

    Chehrehasa F, Meedeniya AC, Dwyer P, Abrahamsen G, Mackay-Sim A (2009) EdU, a new thymidine analogue for labelling proliferating cells in the nervous system. J Neurosci Methods 177(1):122–130. https://doi.org/10.1016/j.jneumeth.2008.10.006

    CAS  PubMed  Article  Google Scholar 

  107. 107.

    Buck SB, Bradford J, Gee KR, Agnew BJ, Clarke ST, Salic A (2008) Detection of S-phase cell cycle progression using 5-ethynyl-2′-deoxyuridine incorporation with click chemistry, an alternative to using 5-bromo-2′-deoxyuridine antibodies. Biotechniques 44(7):927–929. https://doi.org/10.2144/000112812

    CAS  PubMed  Article  Google Scholar 

  108. 108.

    Kelly KJ, Sandoval RM, Dunn KW, Molitoris BA, Dagher PC (2003) A novel method to determine specificity and sensitivity of the TUNEL reaction in the quantitation of apoptosis. Am J Physiol Cell Physiol 284(5):C1309–C1318. https://doi.org/10.1152/ajpcell.00353.2002

    CAS  PubMed  Article  Google Scholar 

  109. 109.

    Mangili F, Cigala C, Santambrogio G (1999) Staining apoptosis in paraffin sections. Advantages and limits. Anal Quant Cytol Histol 21(3):273–276

    CAS  PubMed  Google Scholar 

  110. 110.

    Kyrylkova K, Kyryachenko S, Leid M, Kioussi C (2012) Detection of apoptosis by TUNEL assay. Methods Mol Biol 887:41–47. https://doi.org/10.1007/978-1-61779-860-3_5

    CAS  PubMed  Article  Google Scholar 

  111. 111.

    Huerta S, Goulet EJ, Huerta-Yepez S, Livingston EH (2007) Screening and detection of apoptosis. J Surg Res 139(1):143–156. https://doi.org/10.1016/j.jss.2006.07.034

    CAS  PubMed  Article  Google Scholar 

  112. 112.

    Kanoh M, Takemura G, Misao J, Hayakawa Y, Aoyama T, Nishigaki K, Noda T, Fujiwara T, Fukuda K, Minatoguchi S, Fujiwara H (1999) Significance of myocytes with positive DNA in situ nick end-labeling (TUNEL) in hearts with dilated cardiomyopathy: not apoptosis but DNA repair. Circulation 99(21):2757–2764

    CAS  PubMed  Article  Google Scholar 

  113. 113.

    de Torres C, Munell F, Ferrer I, Reventos J, Macaya A (1997) Identification of necrotic cell death by the TUNEL assay in the hypoxic-ischemic neonatal rat brain. Neurosci Lett 230(1):1–4

    PubMed  Article  Google Scholar 

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Correspondence to Tomáš Roušar.

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Majtnerová, P., Roušar, T. An overview of apoptosis assays detecting DNA fragmentation. Mol Biol Rep 45, 1469–1478 (2018). https://doi.org/10.1007/s11033-018-4258-9

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Keywords

  • Apoptosis
  • DNA fragmentation
  • Apoptosis assays
  • DNA ladder
  • TUNEL assay
  • Comet assay