The use of 7-amino actinomycin D in identifying apoptosis: simplicity of use and broad spectrum of application compared with other techniques.
The detection and quantitation of apoptotic cells is becoming increasingly important in the investigation of the role of apoptosis in cellular proliferation and differentiation. The pathogenesis of hematologic disorders such as aplastic anemia and the development of neoplasia are believed to involve dysregulation of apoptosis. To quantitate accurately the proportion of apoptosis cells within different cell types of a heterogeneous cell population such as blood or bone marrow, a method is required that combines the analysis of large numbers of cells with concurrent immunophenotyping of cell surface antigens. In this study, we have evaluated such a method using the fluorescent DNA binding agent, 7-amino actinomycin D (7AAD), to stain three diverse human cell lines, induced to undergo apoptosis by three different stimuli. Flow cytometric analysis defines three populations on the basis of 7AAD fluorescence and forward light scatter. We have shown by cell sorting and subsequent morphological assessment and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling that the populations defined by 7AAD represent live, apoptotic, and late-apoptotic/dead cells. This method is quick, simple, reproducible, and cheap and will be a valuable tool in the investigation of the role of apoptosis in normal physiology and in disease states.
Philpott
N J
NJ
Division of Haematology, Department of Cellular and Molecular Sciences, St. George's Hospital Medical School, London, UK.
Turner
A J
AJ
Scopes
J
J
Westby
M
M
Marsh
J C
JC
Gordon-Smith
E C
EC
Dalgleish
A G
AG
Gibson
F M
FM
D017209:Apoptosis / Q000187:drug effects; D002166:Camptothecin / Q000494:pharmacology; D002277:Carcinoma / Q000473:pathology; D002452:Cell Count; D002469:Cell Separation; D004249:DNA Damage; D004273:DNA, Neoplasm / Q000032:analysis* / Q000187:drug effects; D003609:Dactinomycin / Q000031:analogs & derivatives*; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute / Q000473:pathology; D015459:Leukemia-Lymphoma, Adult T-Cell / Q000473:pathology; D010190:Pancreatic Neoplasms / Q000473:pathology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D004273:DNA, Neoplasm; D005456:Fluorescent Dyes; D003609:Dactinomycin; C025942:7-aminoactinomycin D; D002166:Camptothecin