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

Figure 9

Phagocytic activity of giant phagocytes (Gϕ) and the effects of NAC on Gϕ phagocytosis. Freshly isolated PMN were exposed for 12 h (29 cycles) or 24 h (56 cycles) to intermittent hypoxia (IH), 24 h sustained hypoxia (SH), or normoxia (N) and then cultured at normoxia for additional six days. NAC (20 μM) was added to PMN cultures 10 min prior to exposing to N, IH, or SH. The developed Gϕ (7 d culture) were incubated for 2 h with carboxylate-modified fluorescent yellow-green latex beads. Cytospins were prepared, fixed, stained with LC3B (red), and analyzed by confocal microscopy (see Materials and Methods). Nuclei were stained with DAPI. (a) The average number of beads/cell in Gϕ developed after exposure to N, IH, or SH without or with NAC in three independent experiments. In each experiment at least 10 cells were counted in each condition. (b) Representative photomicrographs of phagocytosis by Gϕ developed after exposure to N, IH, or SH without (upper panel) or with (lower panel) NAC. (c) A representative analysis of intracellular latex beads localization using 3D reconstructing software IMARIS z-stack for 24 h IH-treated Gϕ in the presence of NAC. Upper panel in (c) represents an image of the Gϕ. Lower panel in (c) represents cross sections (, , and ) for localization of the latex beads.

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