Effect of density gradient material upon ex-vivo neutrophil behaviour, and Effect of neutrophil extracellular traps upon the growth and survival of periodontopathogenic bacteriaud

摘要

Neutrophils isolation requires an efficient and reproducible method to obtain viable cells, and (ex vivo) handling should not influence neutrophil behaviouror or activation. Neutrophil activation relies on nicotinamide adenine dinucleotide phosphate (NADPH) oxidase for the subsequent reactive oxygen species (ROS) production, which is essential for neutrophil defense mechanisms; phagocytosis and neutrophil extracellular traps (NETs). This study investigated the use of Percoll and Histopaque density gradients for isolating neutrophils upon ROS production, NETs, cell viability and metabolic activity. ROS and NET production were analysed in unstimulated, primed and simulated cells. Percoll isolation of neutrophils provided higher cell yields. Histopaque isolated cells demonstrated higher levels of baseline unstimulated ROS production and consistent with that, higher metabolic activity, implying higher baseline activation. Subsequent ROS generation and NET formation in unstimulated cells and following priming and stimulation, were lower for Histopaque than Percoll. Metabolic activity was lower at the point of NET release (180min) for Histopaque than Percoll. In conclusion, the current density gradient method used by the Periodontal Research Group in Birmingham, (Percoll) provided a higher yield, increased sensitivity to neutrophil priming and stimulation, and expressed increased sensitivity to NET production. Percoll will become the gradient material of choice for all future studies involving ROS and/or NET production.