The use of 7-amino actinomycin D in identifying apoptosis: simplicity of use and broad spectrum of application compared with other techniques

Abstract

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.

Evaluation of a Gemcitabine‐Doxorubicin‐Paclitaxel Combination Schedule through Flow Cytometry Assessment of Apoptosis Extent Induced in Human Breast Cancer Cell Lines

“…[12][13][14][15] There are many ways to detect apoptosis: detection of typical morphologic features of the cell population by light or electron microscopy, time-lapse photography, detection of DNA fragmentation by gel electrophoresis, the TUNEL assay, and flow cytometry-based methods (for example, PI staining). In this study, flow cytometric analysis with 7AAD was used 7,8) to determine the extent of drug-induced apoptosis on tumor cell lines. 7AAD stains live, apoptotic, and dead cells differentially because of the altered accessibility of DNA in each subpopulation.…”

“…Survival was expressed as the percentage of viable cells in treated samples relative to nontreated control cells. Analysis of apoptosis by flow cytometry with 7-aminoactinomycin D (7AAD) staining This assay was developed according to the method previously described by Schmid et al 7) and validated by Philpott et al 8) Briefly, 7AAD (Sigma Chemical Company) was dissolved in acetone and diluted in PBS to a concentration of 200 µg/ml. This solution was kept at −20°C and protected from light until use.…”

All‐trans retinoic acid prevents apoptosis of human marrow CD34+ cells deprived of haematopoietic growth factors

“…Moreover, the anti-apoptotic Representative experiment of the effects of ATRA (1 lmol/l) on the apoptosis of marrow CD34 + cells deprived of haematopoietic growth factors. CD34 + cells selected on d 0 were cultured in a serum-free medium for 7 d and the apoptotic status of the CD34 + cells was determined by the analysis of membrane permeability to high doses of 7AAD (Philpott et al, 1996). Effects of ATRA (1 lmol/l) on the apoptosis of marrow CD34 + cells deprived of haematopoietic growth factors.…”

Effects of antithymocyte globulin on bone marrow CD34+ cells in aplastic anaemia and myelodysplasia

“…High concentrations of ATG (equivalent to serum levels found during infusion) inhibited colony growth from CD34 cells. This was con®rmed using 7 amino-actinomycin D (7AAD) staining (Schmid et al, 1994a,b;Philpott et al, 1996), which showed that high ATG concentrations caused the death of normal CD34 cells.…”