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

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 × 10⁶ 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 (NaN₃). LgCL was inhibited by SOD but not by the other agents, whereas LmCL was inhibited by NaN₃ 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.