Analysis of Neutrophil Bactericidal Activity

Abstract

This chapter describes two methods for measuring the bactericidal activity of neutrophils. These are a new simple fluorescence-based assay, which quantifies bactericidal activity by measuring changes in bacterial fluorescence associated with a loss of membrane potential over time, and a more traditional colony counting protocol. Two variations of these techniques are presented: a "one-step" protocol providing a composite measure of phagocytosis and killing, and a "two-step" protocol that allows calculation of separate rate constants for both of these processes.

Peptoanaerobacter stomatis interactions with neutrophils.

“…The generation of ROS is highly damaging to macromolecules such as protein and DNA, therefore resulting strongly bactericidal. Reactive oxygen species may also cause destruction in surrounding tissues, and are capable of inducing immune cell apoptosis (Parker et al, 2014).…”

“…It is composed of two transmembrane proteins (p22phox and gp91phox/ NOX2, which form the cytochrome b 558 ), three cytosolic proteins (p47phox, p67phox, p40phox) and a GTPase (Rac1 or Rac2) (Hurtado-Nedelec et al, 2014). All these components assemble at the membrane upon cell activation to form a functional electron-transfer system, which ferries electrons over the membrane from cytosolic NADPH to O 2 , forming superoxide (O 2 − ) (Parker et al, 2014). Superoxide is then converted to hydrogen peroxide, HOCl -in the presence of neutrophil myeloperoxidase -, or other microbicidal oxidants that synergize with granule proteins to kill internalized microorganisms (Dinauer, 2014).…”

“…neutrophils are also able to produce significant amounts of intracellular ROS even in the absence of phagosome formation (Parker et al, 2014).…”

“…Since 80-85 % of the flavocytochrome b 558, a component of the NADPH oxidase, is localized in the membrane of neutrophils specific granules the intracellular (non-phagosomal) ROS could be explained by the NADPH-oxidase activation in the granule membranes (Parker et al, 2014).…”

“…The biological relevance of granule-localized, non-phagosomal ROS is still not clear, but they likely participate in cell signaling (Parker et al, 2014).…”