Intermittent Hypoxia Affects the Spontaneous Differentiation In Vitro of Human Neutrophils into Long-Lived Giant Phagocytes

Figure 6

PMA-dependent ROS production, and the effects of diphenyl iodide (DPI) (10 μM) and N-acetylcysteine (NAC) (20 μM) on viability and development of giant phagocytes (Gϕ). PMN were cultured during 24 h in normoxia (N), intermittent hypoxia (IH) (56 cycles), or sustained hypoxia (SH) without or with inhibitors and then cultured at normoxia for additional six days. (a) Intracellular ROS production was detected in PMA-activated Gϕ by NBT test (see Materials and Methods). DPI was added to Gϕ 2 h prior to PMA stimulation. Bright-field microscopy of Giemsa-stained cultures in the various oxygen treatments. (b) DPI or NAC were added to PMN cultures 10 min prior to exposing to N, IH, and SH. Then PMN were cultured during the next 6 days at normoxia. Equal volumes of DMSO were added as a negative control. Bright-field microscopy of living cultures in the various treatments. (c) PMN viability was detected immediately after 24 h of the hypoxic treatments in untreated (1), DPI treated (2), or NAC-treated cells (3) using WST-1 assay. The measured absorbance (OD) directly correlates to the number of viable cells in each treatment.

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