Analysis of early apoptotic events in individual cells by fluorescence intensity and polarization measurements

Apoptosis is a dynamic process of variable duration. The ability to continuously detect the death process occurring in single or subgroups of cells is therefore very important in identifying apoptotic cells within a complex population. The Individual Cell Scanner (ICS), a multiparametric, multilaser-based scanning static cytometer, was used in the present report for the continuous monitoring of the apoptosis process. Fluorescence intensity (FI), polarization (FP), kinetic measurements, and cluster analysis of subpopulations were carried out utilizing various fluorescent probes. Hydrogen peroxide-induced apoptosis was monitored online in intact live lymphocytes by continuous sequential measurements of intracellular hyperpolarization. Plasma membrane asymmetry, mitochondrial membrane potential, and lysosomal rupture were monitored in individual cells. Cytoplasmic condensations, due to cell shrinkage and early lysosomal rupture, were found to be very early events of apoptosis. The new analytical capabilities suggested here may provide simple and convenient methods for detecting apoptosis from its earlier stages.