Analysis and assessment of the capacity of neutrophils to produce reactive oxygen species in a 96-well microplate format using lucigenin- and luminol-dependent chemiluminescence

Abstract

The chemiluminescence (CL) assay has been used to measure the reactive oxygen species (ROS)-generating capacity of phagocytes. To achieve more optimal measurement conditions for a multi-channel microplate photon-counting CL analyzer with the cooled charge-coupled device (CCD) camera which offers enhanced sensitivity, we investigated factors affecting the variability in lucigenin-dependent CL (LgCL) measurement of human neutrophils stimulated with either opsonized zymosan (OZ) or phorbol myristate acetate (PMA). We obtained sensitive LgCL responses with good reproducibility and rapid data-acquisition using 50 μl neutrophils (3×10 6 cells/ml) and 50 μl of 0.5 mM lucigenin per well, in addition to either 100 μl of OZ (5 mg/ml) when zymosan was opsonized with 10–20% serum or 100 μl of PMA solution (1×10 −6 M) with automatic regular intervals of mixing and detection during the continuous measurement at 37°C. Furthermore, we studied the contribution of various ROS to LgCL and luminol-dependent CL (LmCL) using modulators of ROS metabolism including superoxide dismutase (SOD), catalase, deferoxamine and sodium azide (NaN 3). LgCL was inhibited by SOD but not by the other agents, whereas LmCL was inhibited by NaN 3 and deferoxamine. Thus, it was demonstrated that LgCL detects the superoxide anion with high selectivity whereas the LmCL assay measures myeloperoxidase (MPO)-mediated formation of hypochlorous acid. Such microplate-based multiple measurements facilitate the accurate assessment of phagocytic function.