Fluorescence microscopy of pneumolysin-treated platelets. (A) Pneumolysin-treated platelets were stained for F-actin (green) and CD62P (magenta). Platelets were not permeabilized, with the exception of the Triton X-100 control. Insets, Single platelets at higher magnification and the line used for measuring fluorescence intensities shown in panel B. In the presence of 3.0 and 30 ng/mL pneumolysin, intracellular staining of CD62P and α-tubulin become visible. At 200 ng/mL pneumolysin, vesicles staining strongly for pneumolysin surround the platelets (compare Figure 1F). (B) Staining pattern of CD62P throughout single cells treated with pneumolysin was quantified to distinguish between cytoplasmic and only surface-associated CD62P staining. The pattern indicates that CD62P is stained intracellularly and not extracellularly. The different concentrations of pneumolysin used are color coded: 3.0 ng/mL (blue), 30 ng/mL (orange), 300 ng/mL (green). (C) Orthogonal views of confocal Z-stacks and 3D isosurface rendering of pneumolysin-treated platelets stained for F-actin (green) and CD62P (magenta). It shows distinct intracellular accumulation of anti-CD62P antibody in platelets treated with different concentration of pneumolysin and membrane permeabilization with Triton X-100 and surface expression of CD62P upon TRAP-6 stimulation.