Activated caspases are localized in the cytoplasm of maturing MKs.

Mobilized blood CD34⁺ cells were cultured as in Figure 1. Panels A-D: labeling with the anti–caspase-3a Ab (red). In panels A-C, a double labeling was performed between the anti–caspase-3a Ab (red) and the anti-VWF Ab (green). DNA was counterstained with DAPI. Cells were examined by confocal microscopy and an overlay representation is shown. Results illustrate a typical section. Round maturing MKs may have either no labeling with the anti–caspase-3 Ab (A) or a granular pattern of staining (B). This granular labeling does not colocalize with the anti-VWF staining. In panel C, an apoptotic MK, as shown by the DAPI labeling, has a diffuse staining by the anti–caspase-3a Ab. In panel D, a double labeling between the anti–caspase-3a Ab (red) and anti-CD63 Ab (green) is illustrated. No colocalization between these 2 labelings was found. Panel E: labeling with an anti–procaspase-3 Ab (green). Two-color immunofluorescence localization of procaspase-3 (green) and VWF (red). Note that procaspase-3 is present in the cytoplasm of proplatelet-forming MKs, including the proplatelets with a diffuse staining pattern. Original magnification, × 1000.