ANALYSIS AND ASSESSMENT OF THE CAPACITY OF NEUTROPHILS TO PRODUCE REACTIVE OXYGEN SPECIES IN A 96-WELL MICROPLATE FORMAT USING LUCIGENIN-DEPENDENT AND LUMINOL-DEPENDENT CHEMILUMINESCENCE

The chemiluminescence (CL) assay has been used to measure the reactive oxygen species (ROS)-generating capacity of phagocytes. To achieve mo re optimal measurement conditions for a multi-channel microplate photo n-counting CL analyzer with the cooled charge-coupled device (CCD) cam era which offers enhanced sensitivity, we investigated factors affecti ng the variability in lucigenin-dependent CL (LgCL) measurement of hum an neutrophil; stimulated with either opsonized zymosan (OZ) or phorbo l myristate acetate (PMA). We obtained sensitive LgCL responses with g ood reproducibility and rapid data-acquisition using 50 mu l neutrophi ls (3 x 10(6) cells/ml) and 50 mu l of 0.5 mM lucigenin per well, in a ddition to either 100 mu l of OZ (5 mg/ml) when zymosan was opsonized with 10-20% serum or; 100 mu l of PMA solution (1 x 10(-6) M) with aut omatic regular intervals of mixing and detection during the continuous measurement at 37 degrees C. Furthermore, we studied the contribution of various ROS to LgCL and luminol-dependent CL (LmCL) using modulato rs of ROS metabolism including superoxide dismutase (SOD), catalase, d eferoxamine and sodium azide (NaN3). LgCL was inhibited by SOD but not by the other agents, whereas LmCL was inhibited by NaN, and deferoxam ine, 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 m ultiple measurements facilitate the accurate assessment of phagocytic function. (C) 1997 Elsevier Science B.V.