Neutrophil Isolation and Cell Death

Human polymorphonuclear cells (PMNs) were freshly isolated from Na-citrate-treated blood of healthy donors. For neutrophil-isolation, dextran-sedimentation and density gradient centrifugation using Ficoll-Paque Plus (Amersham Bioscience) was used according to the manufacturer's instruction. Cell purity was determined by Giemsa staining and was always above 99%. PMNs were suspended to a final density of 1×10⁶ cells/0.5 ml in RPMI 1640 culture medium (PAA Laboratories GmbH) supplemented with 10% heat-inactivated FCS (PAA Laboratories GmbH) and immediately used for the experiments. All incubations were performed at 37°C in humidified air with 5% CO₂.
To measure cell death induction 1x10⁶ cells/0.5 ml PMNs were cultured at 37°C in a 5% CO₂ atmosphere in 24-well plates and bacterial supernatants were added as indicated. After 1 h cell death induction was analyzed as described previously [78 (link),79 (link)].