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
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
Elmore S (2007) Apoptosis: a review of programmed cell death. Toxicol Pathol 35(4):495–516. https://doi.org/10.1080/01926230701320337
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
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
Li J, Yuan J (2008) Caspases in apoptosis and beyond. Oncogene 27(48):6194–6206. https://doi.org/10.1038/onc.2008.297
Mattson MP (2000) Apoptosis in neurodegenerative disorders. Nat Rev Mol Cell Bio 1(2):120–129. https://doi.org/10.1038/35040009
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
Saraste A, Pulkki K (2000) Morphologic and biochemical hallmarks of apoptosis. Cardiovasc Res 45(3):528–537
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
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
Hacker G (2000) The morphology of apoptosis. Cell Tissue Res 301(1):5–17
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
Wyllie AH (1980) Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284(5756):555–556
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
Widlak P (2000) The DFF40/CAD endonuclease and its role in apoptosis. Acta Biochim Pol 47(4):1037–1044
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
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
Nagata S (2000) Apoptotic DNA fragmentation. Exp Cell Res 256(1):12–18. https://doi.org/10.1006/excr.2000.4834
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
Li X, Darzynkiewicz Z (1995) Labelling DNA strand breaks with BrdUTP. Detection of apoptosis and cell proliferation. Cell Proliferat 28(11):571–579
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
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
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
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
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
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
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
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
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
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
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
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
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
Ostling O, Johanson KJ (1984) Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem Biophys Res Commun 123(1):291–298
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
Fairbairn DW, Olive PL, O’Neill KL (1995) The comet assay: a comprehensive review. Mutat Res 339(1):37–59
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
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
Choucroun P, Gillet D, Dorange G, Sawicki B, Dewitte JD (2001) Comet assay and early apoptosis. Mutat Res 478(1–2):89–96
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
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
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
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
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
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
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
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
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
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
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
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
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
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
Mangili F, Cigala C, Santambrogio G (1999) Staining apoptosis in paraffin sections. Advantages and limits. Anal Quant Cytol Histol 21(3):273–276
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
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
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
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
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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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DOI: https://doi.org/10.1007/s11033-018-4258-9