Efficacy and specificity of different methods for human neutrophil extracellular trap isolation and handling

INTRODUCTION: Although in vitro incubation of various cell types with neutrophil extracellular traps (NETs) is commonly used to investigate the influence of NETs on cellular function, it is unclear which human NET isolation and handling protocol is superior. The present study sought to assess the efficacy (yield and purity) and efficiency (time taken) of different available human NET isolation and handling protocols. MATERIAL AND METHODS: Neutrophils isolated from human blood were stimulated using phorbol 12-myristate 13-acetate. Four distinct protocols were used to isolate NETs, and the yield was quantified using fluorimetry. RESULTS: Addition of the restriction enzyme AluI prior to centrifugation is unique to the most effective NET isolation method, yielding a NET concentration of 1077.22 ±229.04 ng/ml (at 523 nm) measured with PicoGreen. Immediate centrifugation to pellet neutrophils is unique to the most efficient method. CONCLUSIONS: Balancing protocol efficacy and efficiency, the method incorporating centrifugation for 5 min at 450 × γ to pellet neutrophils is more than adequate.