Plg enhances phagocytosis of apoptotic thymocytes by J774A.1 cells. J774A.1 cells, a murine macrophage-like cell line, were pretreated with or without Plg (1 μM for 24 hours) and then washed and incubated with fluorescently labeled apoptotic thymocytes. In some experiments, cells were also pretreated with TXA (200 μM) or PI (20 nM) with the Plg. (A) Confocal microscopic images of J77A.1 cells with ingested apoptotic thymocytes labeled with Cell Tracker (green) dye. Images (original magnification, ×63) were captured at room temperature under Leica TCS-SP2-AOBS spectral laser scanning confocal microscope and analyzed using the Image-Pro Plus software. Plasma membrane (red) is marked with Cell Mask plasma membrane stain. The 4′,6 diamidino-2-phenylindole stains nuclei (blue) of both macrophages and apoptotic thymocytes. A zoomed image from Plg-treated cells (white inset) shows breakdown of ingested apoptotic thymocytes (white arrows). (B) Quantification (means ± SD) of IOD of total fluorescence intensity of labeled apoptotic bodies per macrophage nucleus in a microscopic field. (C) Flow cytometry quantification of ingested apoptotic thymocytes labeled with green fluorescence in treated or untreated J774A.1 cells. Bars are ±SD of average MFI of the macrophage population as measured by flow cytometry and analyzed using CellQuest software. Prior to analysis, cell-surface fluorescence was quenched with Trypan blue. Results are representative of 3 independent experiments. AT, apoptotic thymocytes; IOD, integrated optical density; MFI, median fluorescence intensity; PI, plasmin inhibitor D-Val-Phe-Lys chloromethylketone dihydrochloride.