Comparison of comet assay, electron microscopy, and flow cytometry for detection of apoptosis.
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.
Yasuhara
Shingo
S
Department of Anesthesiology & Critical Care, Massachusetts General Hospital, Shriners Hospital for Children, and Harvard Medical School, 55 Fruit Street, Boston, MA 02114, USA.
Zhu
Ying
Y
Matsui
Takashi
T
Tipirneni
Naveen
N
Yasuhara
Yoko
Y
Kaneki
Masao
M
Rosenzweig
Anthony
A
Martyn
J A Jeevendra
JA
D000818:Animals; D017209:Apoptosis; D020552:Comet Assay; D005434:Flow Cytometry; D006801:Humans; D020287:In Situ Nick-End Labeling; D019169:Jurkat Cells; D008854:Microscopy, Electron; D018482:Muscle, Skeletal / Q000166:cytology; D015428:Myocardial Reperfusion Injury / Q000473:pathology; D009206:Myocardium / Q000473:pathology; D009336:Necrosis; D051381:Rats; D017207:Rats, Sprague-Dawley