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

Figure 7

Effects of N-acetylcysteine (NAC) on giant phagocytes (Gϕ) size and LC3B expression. Freshly isolated PMN were exposed for 24 h to intermittent hypoxia (IH, 56 cycles), 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. Equal volumes of DMSO were added as a negative control. Fixed cytospins were stained with rabbit anti-LC3B primary Abs (diluted 1/100) or corresponding isotype controls (rabbit IgG) followed by 1/400 CF 488A anti-rabbit IgG staining (green). Nuclei were stained with DAPI (blue). (a) Area of cells (μm²) in three independent experiments. (b) Fluorescence intensity (FI) of LC3B expression, integrated with Image J Software (see Materials and Methods), in three independent experiments. (c) Representative photomicrographs of Gϕ which developed after exposure to N, IH, or SH without (untreated) or with NAC. Isotype controls: untreated or NAC-treated normoxic-Gϕ.