Neutrophil Activation Markers Evaluation

Different markers of neutrophil activation were measured in every plasma sample obtained during follow-up from the 32 patients studied herein, following the strategy addressed in previous studies [51 (link),52 ,53 (link),54 (link),55 (link),56 (link),57 (link)]. Cell-free DNA (cfDNA; Quant-iT PicoGreen dsDNA kit, Life Technologies, Eugene, OR, USA) and nucleosomes (Cell Death Detection ELISA^PLUS kit, Roche, Mannheim, Germany) were measured as markers of the nuclear content of neutrophils released by neutrophils upon NETosis. Calprotectin (Human Calprotectin ELISA kit, Hycult Biotech, Uden, The Netherlands) was measured as a marker of cytoplasmic content and MPO (Human MPO ELISA kit, Abnova, Taoyuan, Taiwan) as a marker of the content of neutrophil granules, were both released upon neutrophil activation by different mechanisms. In all cases the experiments were performed following the manufacturer’s instructions.