Online citations, reference lists, and bibliographies.
← Back to Search

Comparison Of Comet Assay, Electron Microscopy, And Flow Cytometry For Detection Of Apoptosis

S. Yasuhara, Y. Zhu, T. Matsui, N. Tipirneni, Yoko Yasuhara, M. Kaneki, A. Rosenzweig, J. A. J. Martyn
Published 2003 · Medicine, Biology

Cite This
Download PDF
Analyze on Scholarcy
Share
Differentiating apoptosis from necrosis is a challenge in single cells and in parenchymal tissues. The techniques available, including in situ TUNEL (Terminal deoxyribonucleotide transferase-mediated dUTP-X Nick End-Labeling) staining, DNA ladder assay, and flow cytometry, suffer from low sensitivity or from a high false-positive rate. This study, using a Jurkat cell model, initially evaluated the specificity of the neutral comet assay and flow cytometry compared to the gold standard, electron microscopy, for detection of apoptosis and necrosis. Neutral comet assay distinguished apoptosis from necrosis in Jurkat cells, as evidenced by the increased comet score in apoptotic cells and the almost zero comet score in necrotic cells. These findings were consistent with those of electron microscopy and flow cytometry. Furthermore, using rats with burn or ischemia/reperfusion injury, well-established models of skeletal and cardiac muscle tissue apoptosis, respectively, we applied the comet assay to detect apoptosis in these muscles. Neutral comet assay was able to detect apoptotic changes in both models. In the muscle samples from rats with burn or ischemia-reperfusion injury, the comet score was higher than that of muscle samples from their respective controls. These studies confirm the consistency of the comet assay for detection of apoptosis in single cells and provide evidence for its applicability as an additional method to detect apoptosis in parenchymal cells.
This paper references
Quan - titative differentiation of both free 3  - OH and 5  - OH DNA ends between heat - induced apoptosis and necrosis
T Ikezu (1998)
10.1177/002215549804600909
Quantitative Differentiation of Both Free 3′-OH and 5′-OH DNA Ends Between Heat-induced Apoptosis and Necrosis
R. Hayashi (1998)
10.1002/(SICI)1097-0320(19990201)35:2<145::AID-CYTO6>3.0.CO;2-2
Identification of nuclei from apoptotic, necrotic, and viable lymphoid cells by using multiparameter flow cytometry.
C. Matteucci (1999)
10.1002/1097-0320(20010501)44:1<65::AID-CYTO1083>3.0.CO;2-Q
Oncosis is associated with exposure of phosphatidylserine residues on the outside layer of the plasma membrane: a reconsideration of the specificity of the annexin V/propidium iodide assay.
H. Lecoeur (2001)
10.1016/S0921-8777(97)00013-X
The role of N-acetylcysteine as a putative radioprotective agent on X-ray-induced DNA damage as evaluated by alkaline single-cell gel electrophoresis.
G. Abt (1997)
10.1161/01.CIR.104.3.330
Akt Activation Preserves Cardiac Function and Prevents Injury After Transient Cardiac Ischemia In Vivo
T. Matsui (2001)
10.1007/s004180050402
Early detection of staurosporine-induced apoptosis by comet and annexin V assays
T. Godard (1999)
10.1080/095530098141906
Rejoining of DNA single- and double-strand breaks in human white blood cells exposed to ionizing radiation.
J. Banáth (1998)
10.1385/1-59259-179-5:163
The Comet Assay
A. Collins (2002)
The comet assay. An overview of techniques.
P. L. Olive (2002)
10.1152/AJPENDO.2000.279.5.E1114
Skeletal muscle apoptosis after burns is associated with activation of proapoptotic signals.
S. Yasuhara (2000)
10.1016/0003-2697(90)90180-H
Isolation of transcriptionally active nuclei from striated muscle using Percoll density gradients.
C. G. Hahn (1990)
10.1016/0092-8674(91)90614-5
The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis
N. Itoh (1991)
10.1016/S0027-5107(01)00123-3
Comet assay and early apoptosis.
P. Choucroun (2001)
10.1016/S0091-679X(08)60209-5
Chapter 4 Isolation of Skeletal Muscle Nuclei
L. Kuehl (1977)
10.1043/0003-9985(1999)123<0529:HSPTR>2.0.CO;2
Histologic sectioning produces TUNEL reactivity. A potential cause of false-positive staining.
G. Sloop (1999)
Cell death of AKR-2B fibroblasts after serum removal: a process between apoptosis and necrosis.
A. Simm (1997)
10.1074/jbc.272.40.25289
Analysis of Thermal Injury-induced Insulin Resistance in Rodents
T. Ikezu (1997)
10.1002/(SICI)1096-9861(19970203)378:1<88::AID-CNE5>3.0.CO;2-G
Non‐NMDA and NMDA receptor‐mediated excitotoxic neuronal deaths in adult brain are morphologically distinct: Further evidence for an apoptosis‐necrosis continuum
C. Portera-Cailliau (1997)
10.2307/3578650
Radiation-induced apoptosis measured in TK6 human B lymphoblast cells using the comet assay.
P. L. Olive (1993)
10.1177/002215540004800709
Protease-elicited TUNEL Positivity of Non-apoptotic Fixed Cells
I. Gál (2000)
DNA double-strand breaks measured in individual cells subjected to gel electrophoresis.
P. L. Olive (1991)
10.1016/S0891-5849(02)00967-X
DNA damage and apoptosis in hydrogen peroxide-exposed Jurkat cells: bolus addition versus continuous generation of H2O2
A. Barbouti (2002)
10.1083/JCB.27.2.365
ISOLATION OF SKELETAL MUSCLE NUCLEI
J. C. Edelman (1965)
10.1006/EXCR.1995.1348
Sizing highly fragmented DNA in individual apoptotic cells using the comet assay and a DNA crosslinking agent.
P. L. Olive (1995)
DNA damage and apoptosis in hydrogen peroxide-exposed Jurkat cells: bolus addition versus continuous generation of H(2)O(2).
A. Barbouti (2002)
10.1002/(SICI)1097-0320(19991101)37:3<197::AID-CYTO6>3.0.CO;2-L
Annexin V used for measuring apoptosis in the early events of cellular cytotoxicity.
J. Aubry (1999)
The comet assay. Principles, applications, and limitations.
A. Collins (2002)
10.1097/00004647-199704000-00003
Detection of Higher-Order 50- and 10-kbp DNA Fragments before Apoptotic Internucleosomal Cleavage after Transient Cerebral Ischemia
J. P. Macmanus (1997)
10.1093/NAR/20.19.5243
A simple and rapid method for detection of apoptosis in human cells.
F. Roesl (1992)
10.1111/j.1365-2249.1991.tb05639.x
Programmed cell death (apoptosis) in lymphoid and myeloid cell lines during zinc deficiency
S. Martin (1991)
10.1097/00004630-199920060-00007
The 1999 Moyer award. Burn injury induces skeletal muscle apoptosis and the activation of caspase pathways in rats.
S. Yasuhara (1999)
10.1016/0006-291X(84)90411-X
Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells.
O. Ostling (1984)
10.1385/1-59259-179-5:195
Ultrasensitive detection of DNA damage by the combination of the comet and TUNEL assays.
A. Kindzelskii (2002)
10.1016/S0074-7696(08)62312-8
Cell death: the significance of apoptosis.
A. Wyllie (1980)
10.1385/1-59259-179-5:179
The Comet Assay
P. L. Olive (2002)
10.1067/MNC.2000.103724
Cardiac signal transduction
K. Lee (2000)



This paper is referenced by
The influence of photodynamic therapy on apoptosis in human melanoma cell line.
J. Saczko (2005)
10.1002/tox.20373
Monitoring of DNA breakage in embryonic stages of the African catfish Clarias gariepinus (Burchell, 1822) after exposure to lead nitrate using alkaline comet assay
A. Osman (2008)
10.3109/09553002.2012.711502
X-irradiation induces ER stress, apoptosis, and senescence in pulmonary artery endothelial cells
R. A. M. Panganiban (2013)
10.1016/B978-0-12-374912-3.00008-0
Analytical capabilities of the ImageStream cytometer.
E. Zuba-Surma (2011)
Potent reducing effects of vitamin D3 on the frequency of apoptosis induced by arsenic trioxide in NB4 cell line.
H. Mozdarani (2010)
Estudi d'un procés de mort cel·lular induït per una activació no canònica de les caspases caracteritzat per la presència de morfologies necròtiques
Unitat de Bioquímica (2013)
10.7282/T36M3651
Developing a time-dynamic, label-free assay for apoptosis based on optical Gabor filtering
Robert M. Pasternack (2011)
10.1152/ajpendo.00078.2013
Title efficacy of phosphodiesterase 5 inhibitor on distant burn-induced muscle autophagy, microcirculation, and survival rate.
Sachiko Hosokawa (2013)
Pese a que se evidenciaron dificultades para establecer la relación in vivo de estos contaminantes ambientales con respecto a los efectos genotóxicos inducidos en los
Leidy Ortegon-Torres (2014)
10.1039/c3an02205f
A novel strategy for real-time and in situ detection of cytochrome c and caspase-9 in Hela cells during apoptosis.
Qingqing Wen (2014)
10.1002/cyto.a.20071
Single‐cell gel electrophoresis assay monitors precise kinetics of DNA fragmentation induced during programmed cell death
S. Chandna (2004)
10.18725/OPARU-2818
Prävention der ischämischen Querschnittlähmung durch ein carbamyliertes Fusionsprotein aus Erythropoetin und einem humanen Fc-Fragment bei Aortenokklusion am Schweinemodell
T. Krämer (2013)
MECHANISMS BY WHICH HYPERBARIC OXYGEN THERAPY MAY RESOLVE INFLAMMATION IN CHRONIC WOUNDS
Anwar Jasib Thaaban Almzaiel (2013)
Targeting prostate cancer cell proliferation, stemness and metastatic potential using Costus speciosus derived phytochemicals.
A. Elkady (2019)
10.7150/thno.16141
X-Ray Induced Photodynamic Therapy: A Combination of Radiotherapy and Photodynamic Therapy
G. Wang (2016)
10.1210/JC.2004-0969
All-trans-retinoic acid treatment inhibits the growth of retinoic acid receptor beta messenger ribonucleic acid expressing thyroid cancer cell lines but does not reinduce the expression of thyroid-specific genes.
R. Elisei (2005)
10.1177/1934578X1400900904
Zerumbone Induces G2/M Cell Cycle Arrest and Apoptosis via Mitochondrial Pathway in Jurkat cell Line
H. Rahman (2014)
10.1177/0960327112446816
Optimized noninvasive procedures to measure DNA damage in comet assay
Y. T. Szeto (2012)
10.1016/J.MRGENTOX.2006.11.007
Measurements of DNA damage on silver stained comets using free Internet software.
O. García (2007)
The use of different blood sample preparations in the development of biomarkers for the environmental monitoring of domestic farm animals
David S. Groeger (2014)
10.1007/s10565-008-9072-z
Comet assay: a reliable tool for the assessment of DNA damage in different models
Alok Dhawan (2008)
10.1007/s00432-020-03213-x
Selected ellipticine derivatives, known to target topoisomerase II, suppress the alternative lengthening of telomere (ALT) pathway in telomerase–negative cells
S. Zencir (2020)
10.1152/ajplung.00129.2007
Prostacyclin analogs stimulate VEGF production from human lung fibroblasts in culture.
K. Kamio (2008)
10.1016/j.ejmech.2013.08.055
Synthesis and antitumor activities of novel thiourea α-aminophosphonates from dehydroabietic acid.
X. Huang (2013)
Oncolytic effect of Newcastle disease virus AF2240 strain on the MCF-7 breast cancer cell line.
F. Othman (2010)
10.1155/2014/260210
Effects of Crude Extracts from Medicinal Herbs Rhazya stricta and Zingiber officinale on Growth and Proliferation of Human Brain Cancer Cell Line In Vitro
A. Elkady (2014)
10.1007/s10630-005-0151-5
Prooxidant and Cytotoxic Effects of Thiols Combined with Vitamin B12b
M. E. Solov’eva (2005)
10.1016/j.mrgentox.2010.11.011
Visual estimation of the percentage of DNA in the tail in the comet assay: evaluation of different approaches in an intercomparison exercise.
O. García (2011)
10.1016/j.mito.2013.02.005
Predicted ionisation in mitochondria and observed acute changes in the mitochondrial transcriptome after gamma irradiation: a Monte Carlo simulation and quantitative PCR study.
W. W. Kam (2013)
10.1111/andr.12429
Evaluation of sperm DNA quality in men presenting with testicular cancer and lymphoma using alkaline and neutral Comet assays
K. Kumar (2018)
1-amino-4-phenyl-1,2,3,6-tetrahydropyridine and 1-amino-4-phenylpyridinium salt, the 1-amino analogues of neurotoxins, MPTP and MPP+, induce apoptosis in PC12 cells: detection of apoptotic cells by Comet assay and flow cytometric analysis.
Y. Noda (2004)
Nile Encephalitis Virus Infection and Injury of Neurons by West
B. Shrestha (2003)
See more
Semantic Scholar Logo Some data provided by SemanticScholar