The correlativity analysis of six methods of detecting apoptosis.
The aim of this study was to compare six methods of detecting apoptosis induced by extracellular adenosine triphosphate (ATP) in human leukemic lymphocytes with purinergic P2Z receptors. These methods used were electron microscopy (EM), detection of internucleosomal DNA fragmentation by agarose gel electrophoresis, autoradiographic analysis of DNA fragmentation, in situ labeling of DNA strand breaks with fluorescein dUTP and exogenous terminal deoxynucleotidyl transferase (TUNEL), quantitation of 3'-ends of DNA breaks by labeling with alpha32PdCTP(TdT assay), and quantitation of apoptotic cells with fluorescein-annexin V using flow cytometry (FCA). We found EM and detection of DNA ladder pattern by agarose gel electrophoresis to b specific, but lacking in sensitivity. The combination of autoradiography and gel electrophoresis gave an increase in sensitivity of at least 50 fold although, of all the methods, the TdT assay was shown to be most sensitive. The four methods for quantifying apoptosis-EM, FCA, TUNEL and TdT assay proved to be reliable and gave statistically similar results on apoptotic lymphocytes. These observations indicate it is essential to combine specific, sensitive and quantitative techniques in detecting apoptosis.
Liming
P
P
Department of Laboratory Medicine, First University Hospital, West China University of Medical Sciences, Chengdu 610041.
Bradley
C J
CJ
Liu
J J
JJ
D000255:Adenosine Triphosphate / Q000494:pharmacology; D017209:Apoptosis; D004249:DNA Damage; D004253:DNA Nucleotidylexotransferase / Q000032:analysis*; D005434:Flow Cytometry; D006801:Humans; D020287:In Situ Nick-End Labeling; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000473:pathology*; D018048:Receptors, Purinergic P2 / Q000502:physiology; D014407:Tumor Cells, Cultured
D018048:Receptors, Purinergic P2; D000255:Adenosine Triphosphate; D004253:DNA Nucleotidylexotransferase