Effect of hypoxia on eosinophil survival. Eosinophils were cultured in medium alone or supplemented with either GM-CSF, IL-3 or IL-5 (20 ng/ml) in normoxia (solid columns) or hypoxia (stripped columns) for 24 h, 48 h and 72 h. (A) Flow cytometry analysis of PI stained cells was performed and viable eosinophils were identified as PI-. (B) Eosinophils were cultured in medium in hypoxia or normoxia for 24 h, 48 h and 72 h and stained with AnnexinV and PI. AnnexinV+/PI- cells were gated as viable apoptotic eosinophils; AnnexinV-/PI- were gated as viable cells. (C) Eosinophils were cultured o.n. in medium in normoxia or hypoxia and Bax and Bcl-XL expression was analyzed by Western blot after o.n. incubation (C). Representative blot of three experiments performed on three different eosinophil donors (left panel), and average of densitometry quantification of the three experiments (right panel) are shown.