Koren, E.; Foehr, E.; Faruqi, T.; Gardiner, E.; Mahadevan, R.

Functionally competent, highly purified neutrophils were isolated from healthy human donors. Neutrophil necroptosis and neutrophil extracellular trap (NET) formation were induced using TNF-a in combination with the pan-caspase inhibitor zVAD-FMK and the IAP antagonist BV6. NETs were visualized, quantified, and morphologically characterized by fluorescence microscopy following staining with Hoechst 33342 and Sytox Green. Levels of extracellular, cell-free neutrophil elastase (NE), myeloperoxidase (MPO), and DNA were also measured as indicators of NET release. The ability of TACT507, a proprietary RIPK3 antagonist, to efficiently block NETs formation and NET related necroptosis was evaluated. TACT507 demonstrated concentration dependent, significant inhibition of neutrophil necroptosis and NETs formation.