Relocalization of Bax to the mitochondria in PMNs.

PMNs were cultured as described in Figure 1. Then the cells were stained with 1 μM MitoTracker Green FM, fixed, permeabilized, stained with antibody specific for Bax, and were analyzed with CLSM. (A) In fresh PMN Bax and the mitochondria were localized separately. (B) Overnight-cultured untreated cells demonstrated fusion of Bax and mitochondria into large aggregates (right panel in yellow). (C) Addition of G-CSF preserved the separate localization of Bax and mitochondria. (D) zVAD-fmk did not prevent aggregate formation and colocalization of Bax and mitochondria. (Comment: due to the fixation and permeabilization procedures, the mitochondrial staining [green] showed a more diffuse cytoplasmic pattern than the tubular structures shown in Figure 2, panels A and C). Bar is 5 μm. This figure is representative of at least 4 independent experiments.