A comparison of three flow cytometry methods for evaluating mitochondrial damage during staurosporine-induced apoptosis in Jurkat cells.

Measuring cytochrome c release during apoptosis provides valuable information about the nature and extent of apoptosis. Several years ago a flow cytometric method (based on selective permeabilization of the plasma membrane with digitonin) was developed that has advantages over other techniques. These experiments describe a comprehensive evaluation of that method. Apoptosis was triggered in Jurkat cells with staurosporine and then flow cytometry was used to measure three aspects of mitochondrial damage: (1) cytochrome c release (with the digitonin assay and a commercially available kit based on the same principle), using a DNA-binding dye to define cell cycle stage; (2) loss of mitochondrial cardiolipin, assessed by a decrease in 10 N-nonyl acridine orange (NAO) binding; and (3) loss of mitochondrial membrane potential, assessed by a decrease in tetramethylrhodamineethylester (TMRE) binding. The results from these three assays were compared with an antibody-based assay for cleaved caspase 3. The digitonin assay and the commercially available kit gave comparable results, showing that staurosporine caused cytochrome c release in all phases of the cell cycle and clearly defining those cells that had lost DNA due to internucleosomal DNA fragmentation. The pattern of fluorescence demonstrated that the mitochondrial apoptotic pathway was either the sole or the predominant pathway to be activated and that cytochrome c release in an individual cell was all-or-nothing. However, comparison with the other assays showed that the cytochrome c release assay underestimated the true extent of apoptosis. This was caused by the selective loss of some digitonin-treated apoptotic cells. The flow cytometry assay for cytochrome c release provides valuable information but it underestimates the percentage of apoptotic cells.

High-affinity binding sites for heparin generated on leukocytes during apoptosis arise from nuclear structures segregated during cell death.

During cell death of human cultured leukocytes (Jurkat, HL-60, THP-1, U937) and freshly prepared leukocytes, we observed a greater than 100-fold increase in the affinity of apoptotic and necrotic cells for fluorescein isothiocyanate (FITC)-heparin in comparison with live cells. Binding of FITC-heparin was reversed in the presence of high ionic strength, unlabeled heparan sulfate, and heparin and pentosan polysulfate, but not in the presence of chondroitin and dermatan sulfates. During the course of cell death, the increase in the percentage of cells positive for annexin V binding correlated with the increase in the population positive for binding FITC-heparin. Confocal microscopy demonstrated that heparin binding to dead cells was restricted to 1 or 2 small domains on the surfaces of apoptotic cells and to larger, but still discrete, areas that did not localize with chromatin on ruptured necrotic cells. The heparin-binding domains originated from the nucleus and may correspond to the ribonucleoprotein-containing structures that have recently been shown to segregate within the nucleus of cells and to move onto the cell membrane. We observed that phagocytosis of dead Jurkat cells by monocyte-derived macrophages was blocked when the heparin-binding capacity of the dead cells was saturated by the addition of pentosan polysulfate. From this we concluded that the ability of dead cells to bind to heparan sulfate proteoglycans on the surfaces of macrophages may assist in phagocytic clearance.