Degradation of neutrophil extracellular traps (NETs) by fetal calf serum (FCS). (A) Quantification of percentage of NET-DNA (using Picogreen [Invitrogen] as previously described⁵ ) released by human blood–derived neutrophils (isolated using PolymorphPrep system [Axis-Shield] as recommended by the manufacturer) after stimulation with 25nM PMA for 3 hours at 37°C and 5% CO₂ in the presence of different concentration with FCS. Data are mean and SEM of 3 independent experiments. Significant differences analyzed by unpaired t test. (B) Activity of RPMI medium (Invitrogen) containing 10% FCS (Invitrogen), mouse serum (MS), human serum (HS), or human plasma (HP) in degrading 150 μg/mL calf thymus DNA (Sigma) as determined by agarose gel electrophoresis. Serum or plasma was heat-inactivated at 56°C or 70°C for 30 minutes before experiments. Note that medium containing 56°C heat-inactivated serum or plasma showed degradation of DNA similar to micrococcal nuclease used as a positive control. In contrast, heat inactivation of serum or plasma at 70°C completely abolished this nuclease activity. (C) Confocal immunofluorescence microscopy to visualize degradation of NETs by FCS. Human blood–derived neutrophils were stimulated with 25nM PMA for 2 hours at 37°C and 5% CO₂ in serum-free RPMI to release NETs. Then, 10% FCS heat-inactivated at either 56°C or 70°C was added to the medium for an additional hour. As control, NETs were degraded by adding 500 mU/mL micrococcal nuclease (Worthington Biochemical Corporation) to the medium. NETs were visualized by immunofluorescence microscopy using a rabbit anti myeloperoxidase-antibody (1:300; 1 hour at room temperature; Dako), followed by a secondary Alexa 488–labeled goat anti–rabbit antibody (1:500; 1 hour at room temperature; Invitrogen); samples were embedded in ProlongGold+Dapi (Invitrogen) to counterstain nucleus and extracellular DNA in blue. Mounted samples were examined using an inverted confocal laser-scanning 2-photon microscope Olympus Fluoview FV1000 with Fluoview TM Spectral Scanning technology (Olympus) and a 20×/0.75 UPlanSApo Olympus objective. Note that addition of 56°C heat-inactivated FCS to the cells, in contrast to FCS heat-inactivated at 70°C, resulted in degradation of NETs. Fixation of cells with 4% paraformaldehyde (PFA) for 15 minutes at room temperature did not prevent NET degradation by 56°C heat-inactivated FCS.