The use of Apostain in identifying early apoptosis

Irradiated human peripheral blood lymphoid cells undergo apoptosis and progressively exhibit typical changes in light scatter and plasma membrane integrity that can be easily tracked by flow cytometry. Using this model, we assessed the capacity of a newly developed fluorochrome, Apostain, in identifying early apoptosis in unfixed samples. This probe is a plasma membrane permeant DNA dye that can be conveniently excited at 488 nm and has an emission wavelength > 650 nm. To identify apoptotic cells, Apostain relies on the transient changes of chromatin texture that allow to accommodate more of a DNA dye occurring in early apoptosis. As early as 4 h after irradiation a proportion of cells showed an enhanced Apostain uptake. Consistent with their initial apoptotic nature, these cells had a still integer plasma membrane, as assessed by ethidium bromide, and unaltered light scatter. With time, cells showing the enhanced Apostain uptake started to bind dimly Annexin-V and, later, reduced their forward scatter. After 18 h from irradiation, cells exhibiting a reduced forward scatter exhibited a bright staining with Annexin-V with a concomitant reduction in Apostain uptake, reflecting the gross chromatin disruption characterising the endpoint of apoptosis.