Plasminogen binding is stimulated on early apoptotic cells. U937 cells were either untreated or incubated with 30 ng/mL TNFα or 50 μg/mL cycloheximide for 18 hours at 37°C. To identify early apoptotic cells that specifically bound plasminogen, 3-color FACS analysis was performed with annexin V–phycoerythrin, PI, and FITC-plasminogen (0.2 μM) in the presence or absence of EACA (0.2 M). The cells were gated for size (forward scatter versus PI), and 3 nonoverlapping subpopulations were identified: viable, early apoptotic, and late apoptotic/necrotic. The early apoptotic cells were gated out and analyzed for FITC-plasminogen binding in the presence or absence of EACA, and only the early apoptotic cells are shown here. Because TNFα- and CHX-induced cells resulted in a larger population of early apoptotic cells, there are varying numbers of cells in each condition, but the total number of cells initially analyzed was 20 000. The percentage of early apoptotic cells in each quadrant is shown. MFI indicates mean fluorescence intensity.