Samples were incubated sellckchem with dextran (3%) for 45 min. PMN cells in the supernatant were separated by gradient density centrifugation (250 g, 25 min) with Ficoll-Paque? Plus. Following red blood cell lysis, neutrophils were washed (HBSS without Ca2+ or Mg2+) and re-suspended in complete RPMI medium. To induce spontaneous apoptosis, neutrophils were cultured at 37��C for 24 h (5��106 cells/ml in cultured medium without serum in a humidified atmosphere containing 5% CO2). Purity of isolation and apoptosis of neutrophils were assessed by Wright��s Giemsa staining. Neutrophil preparations with more than 90% apoptotic cells were labeled with 5-(and-6)-carboxy fluoresceindiacetatesuccinimidyl ester (CFSE) (Invitrogen Life Technologies, Barcelona, Spain) following the manufacturer��s instructions.

Labelled cells were used as targets in the phagocytosis assay. Phagocytosis assay Following a 4 h-period of normoxic or hypoxic conditions, macrophages were co-cultured with CFSE-labeled apoptotic neutrophils at a phagocyte-to-target ratio of 110. One hour later, cells were washed thoroughly with PBS. U937 cells were stained with Hoechst 33342 (Sigma-Aldrich, Steinheim, Germany) in order to visualize the nuclei and with a fluorescent mitochondrion-selective probe (MitoTracker? Red 580, Invitrogen Life Technologies, Barcelona, Spain) in order to define the cytoplasm area, and were then fixed with paraformaldehyde 4% for 10 min. Samples were analyzed with a fluorescent microscope (IX81, Olympus, Hamburg, Germany) and the CFSE fluorescent signal was quantified using the static cytometer software ��Scan�� version 2.

03.2 (Olympus, Hamburg, Germany). This system automatically counts the total number of cell nuclei per field and the number of phagocytic cells (green fluorescence on red fluorescence). All treatments were performed in duplicate in 12-well plates, and 20 images per well (around 2000 cells) were recorded. Results are expressed as intensity of fluorescence in arbitrary units. Protein extraction and western blot analysis of HIF-1��, CD36 and TSP-1 expression U937cells (2.5?106 cells) were suspended and incubated on ice for 15 min with 50 ��l of lysis buffer (10 mM HEPES, pH 7.5, 2 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 10 mM NaCl, 1 mM DTT, 10 mM NaF, 0.1 mM Na3VO4, 0.2% NP40, 1 mM Pefabloc SC (AEBSF) (Roche Diagnostics GmbH, Mannheim, Germany), supplemented with Protease Inhibitor Cocktail (Roche Diagnostics GmbH, Mannheim, Germany).

Lysates were centrifuged for 10 min at 4��C (16000 g). Supernatants were considered cytosolic extracts. Pellets were sonicated for 10 min in 50 ��l nuclear extraction buffer (25 mM HEPES, AV-951 pH7.5, 500 mM NaCl, 1 mM DTT, 10 mM NaF, 10% Glycerol, 0,2% NP40, 5 mM MgCl2, 1 mM Pefabloc SC (Roche Diagnostics GmbH, Mannheim, Germany) supplemented with Protease Inhibitor Cocktail (Roche Diagnostics GmbH, Mannheim, Germany).