Measurement of Apoptosis by Staining with 7-Amino-Actinomycin D with Concurrent Dual Color Immunofluorescence by Single Laser Flow Cytometry

ABSTRACT

Apoptosis is a crucial mechanism for the selective elimination of mammalian cells and is involved in many physiological and pathological processes. The heightened awareness of the importance of apoptosis has increased the need for rapid and quantitative methods for measurement of apoptotic changes. Recently, 7-amino-actinomycin D (7-AAD) has been introduced as a valuable fluorescent dye for assessing apoptosis by flow cytometry. When the cells are stained with 7-AAD in the concentration of 10 - 20 ug/ml, live cells are not stained (7-AAD ) and early apoptotic cells are weakly stained (7-AAD ) while late apoptotic or dead cells are stained brightly (7-AAD). On scattergram of forward angle light scatter vs. 7-AAD fluorescence, the three populations can be discriminated not only between each other but also from cell debris or clumps. The apoptotic cells, defined as 7- AAD cells, were demonstrated as apoptotic by morphological observation of the sorted cells. The 7-AAD cell fraction was also demonstrated to be parallel with subdiploid fraction of cells stained with PL However, 7-AAD cells, whose definition is based on the alteration of membrane integrity, have never been demonstrated to be subdiploid fraction by simultaneous DNA staining. Here we directly demonstrate that 7-AAD cells, defined on the scattergram of forward angle light scatter vs. 7-AAD fluorescence, are subdiploid fraction by staining with DNA dye whose fluorescence is collected after 530/30 band pass filter (FL-1). We also demonstrate the effects of 7-AAD concentration, fixation of cells, and proliferation of cells, on the fluorescence pattern, for reference during assessment of apoptosis by simple and rapid method for flow cytometric analysis of cells stained with 7- AAD. We also present a flow cytometric analysis of cells stained with 7-AAD Eor sequential change in apoptotic fraction, with concurrent dual-color immunophenotyping.