Journal: Oxidative Medicine and Cellular Longevity

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

doi: 10.1155/2016/9636937

Figure Lengend Snippet: Effects of hypoxia on giant phagocytes (G ϕ ) area and markers.

Article Snippet: For intracellular staining, cells were permeabilized with 0.5% Triton X-100 (Sigma-Aldrich, Israel) in PBS, at room temperature for 10 min. After blocking with 10% normal goat serum in RPMI-1640, cells were incubated overnight at 4°C using the following primary Abs (dilution 1 : 100) or the corresponding isotype controls: mouse monoclonal anti-CD66b Abs (80H3, AbD Serotec, Oxford, UK) and anti-cytochrome b-245 light chain (p22- phox identification, Clone 44.1, BioLegend, San Diego, CA), rabbit polyclonal anti-neutrophil elastase (NE) (Calbiochem, San Diego, CA), anti-LC3B Abs (Sigma, Israel), and anti-Nox2/gp91- phox Abs (ab131083, Abcam, UK).

Techniques:

Journal: Oxidative Medicine and Cellular Longevity

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

doi: 10.1155/2016/9636937

Figure Lengend Snippet: Effects of hypoxia on gp91- phox and p22- phox expression in giant phagocytes (G ϕ ). 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. For double immunofluorescence staining fixed cytospins were stained with rabbit anti-gp91- phox and mouse anti-p22- phox primary Abs (1/100) or the corresponding isotype controls (rabbit IgG and mouse IgG2) followed by 1/400 CF 488A goat anti-rabbit IgG (green) and CF 647 goat anti-mouse IgG (red) staining. Nuclei were stained with DAPI (blue). Representative data out of 3 independent experiments.

Article Snippet: For intracellular staining, cells were permeabilized with 0.5% Triton X-100 (Sigma-Aldrich, Israel) in PBS, at room temperature for 10 min. After blocking with 10% normal goat serum in RPMI-1640, cells were incubated overnight at 4°C using the following primary Abs (dilution 1 : 100) or the corresponding isotype controls: mouse monoclonal anti-CD66b Abs (80H3, AbD Serotec, Oxford, UK) and anti-cytochrome b-245 light chain (p22- phox identification, Clone 44.1, BioLegend, San Diego, CA), rabbit polyclonal anti-neutrophil elastase (NE) (Calbiochem, San Diego, CA), anti-LC3B Abs (Sigma, Israel), and anti-Nox2/gp91- phox Abs (ab131083, Abcam, UK).

Techniques: Expressing, Isolation, Cell Culture, Double Immunofluorescence Staining, Staining

Journal: Oxidative Medicine and Cellular Longevity

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

doi: 10.1155/2016/9636937

Figure Lengend Snippet: Expression of NADPH oxidase subunits in giant phagocytes (G ϕ ) and the effects of NAC on their 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 μ M) 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. Cytospins were prepared and analyzed by confocal microscopy (see Materials and Methods). The developed G ϕ were stained by double immunofluorescence: (a) for CD66b (red) and gp91- phox (green) in untreated and NAC-treated G ϕ and (b) for gp91- phox (green) and p22- phox (red) in untreated and NAC-treated G ϕ . Nuclei were stained with DAPI. Representative photomicrographs out of 3 independent experiments.

Article Snippet: For intracellular staining, cells were permeabilized with 0.5% Triton X-100 (Sigma-Aldrich, Israel) in PBS, at room temperature for 10 min. After blocking with 10% normal goat serum in RPMI-1640, cells were incubated overnight at 4°C using the following primary Abs (dilution 1 : 100) or the corresponding isotype controls: mouse monoclonal anti-CD66b Abs (80H3, AbD Serotec, Oxford, UK) and anti-cytochrome b-245 light chain (p22- phox identification, Clone 44.1, BioLegend, San Diego, CA), rabbit polyclonal anti-neutrophil elastase (NE) (Calbiochem, San Diego, CA), anti-LC3B Abs (Sigma, Israel), and anti-Nox2/gp91- phox Abs (ab131083, Abcam, UK).

Techniques: Expressing, Isolation, Cell Culture, Negative Control, Confocal Microscopy, Staining, Immunofluorescence