Bax expression and co-localization with mitochondria in neutrophils exposed to various oxygen conditions. Bax expression and co-localization with mitochondria were analyzed by confocal laser scanning microscopy as described in methods. The cytoplasmic distribution of Bax (green fluorescence) and mitochondria (red fluorescence) was studied by double immunofluorescence labeling. (A) A representative photomicrograph of an apoptotic neutrophil with a very high Bax expression (140,000 arbitrary units) and intensive fusion of Bax with mitochondria (yellow), Manders Overlap Coefficient (MOC)>0.6. (B) A pre-apoptotic neutrophil with intensive Bax expression (17,000 arbitrary units). Bax is translocated and co-localized with mitochondria (yellow and orange), MOC>0.6 representing neutrophils predominantly seen at normoxia (Nox). (C) A pre-apoptotic neutrophil with low Bax expression (9,500 arbitrary units). Bax (green) and mitochondria (red) are located separately (MOC<0.6), representing neutrophils predominantly seen in intermittent hypoxia (IH) and sustained hypoxia (SH). (D). The percentage of pre-apoptotic neutrophils with fusion of Bax and mitochondria (MOC>0.6) in Nox, IH and SH. Values represent the means ± standard deviations (n=10). P values represent significance of IH or SH vs. Nox. (E) Relative percentage of Bax expression, assessed by confocal laser scanning microscopy (n=10). The average fluorescence intensity unit per cell, detected by immunofluorescent quantitation at normoxia was considered as baseline (100%), and the effects of hypoxia were calculated as relative decrease of Bax expression. P values represent significance of IH or SH vs. Nox. (F-H) Representative images of Bax expression (green) out of ten experiments in Nox (F), IH (G), and SH (H).