FIG. 4.

Conditioned medium from toxoplasma-infected fibroblasts mediates delayed spontaneous neutrophil apoptosis. Neutrophils were incubated with medium (A), with a 1:4 dilution of conditioned medium from uninfected fibroblasts, with a 1:4 dilution of conditioned medium from infected fibroblasts (B), and with a volume of egressed tachyzoites that had been washed four times, equivalent to a 1:4 dilution of conditioned medium from infected fibroblasts (see Materials and Methods). On the next day, washed cells were stained with GFP-annexin and PI and examined by flow cytometry. A gate was set on the neutrophil population of interest (see top, FSC versus SSC plots), and apoptosis was determined by dual color analysis of these cells (see bottom dot plots of GFP-annexin [FL1-H] versus PI [FL2-H]). Numbers show the percentage of cells within each quadrant. Plots of both FSC versus SSC and FL1-H versus FL2-H for conditioned medium from uninfected fibroblasts and washed tachyzoites were identical to that of medium (A). (C) Percent neutrophils in early apoptosis measured as the percentage of neutrophils in the lower right quadrant of the FL1-H versus FL2-H plots. Results in panels A and B are representative of three experiments. Results in panel C are the means ± standard deviations for triplicate samples and are also representative of three experiments.