Neutrophil activation by immobilized immune complexes was achieved by plating the cells on the immune complex-coated surfaces without any additional stimulus. Other routes of cell activation (CB+fMLP, TNF on fibrinogen, immobilized anti-CD18 antibodies, PMA) were performed as described (54 (link), 55 (link), 58 (link), 59 (link)). For respiratory burst and degranulation assays, 1×105 human or 4×105 murine neutrophils/well were used. Release of superoxide was determined by a cytochrome c reduction test as described (59 (link)). Unless otherwise stated, unstimulated control values were subtracted from the stimulated superoxide release. Degranulation of gelatinase (a marker of the specific and gelatinase granules) was determined by an in-gel gelatinase zymography assay. Neutrophil supernatants collected after 30 min stimulation were centrifuged to remove any remaining cells, supplemented with 4× concentrated nonreducing Laemmli sample buffer and run on an 8% SDS-polyacrilamide gel containing 0.1% gelatin (Sigma). Gels were renatured in 2.5% Triton X-100 and incubated overnight at 37 °C in 200 mM NaCl, 5 mM CaCl2, 50 mM Tris, pH 7.4. Digestion of gelatin pre-polymerized into the gel was visualized by Coomassie Blue staining.
For microscopic observations, 2×106 human or 5×106 murine neutrophils in 1 ml assay medium were plated on immune complex-covered 3.5-cm tissue culture dishes. After 20 min of incubation, the cells were cooled and fixed by the addition of 100 μl formalin (Sigma) to the assay medium. Nonadherent cells were allowed to settle and images were taken on a Leica (Wetzlar, Germany) DMI 6000B inverted microscope with a 20× phase contrast objective, connected to a Leica DFC480 CCD camera.
For microscopic observations, 2×106 human or 5×106 murine neutrophils in 1 ml assay medium were plated on immune complex-covered 3.5-cm tissue culture dishes. After 20 min of incubation, the cells were cooled and fixed by the addition of 100 μl formalin (Sigma) to the assay medium. Nonadherent cells were allowed to settle and images were taken on a Leica (Wetzlar, Germany) DMI 6000B inverted microscope with a 20× phase contrast objective, connected to a Leica DFC480 CCD camera.
