Validation of different chemilumigenic substrates for detecting extracellular generation of reactive oxygen species by phagocytes and endothelial cells

Chemiluminescence is a widely used tool to detect extracellular generation of reactive oxygen species (ROS). In the present study we tested four different chemilumigenic substrates (CLS)-luminol, isoluminol, lucigenin and pholasin-to detect extracellular CL in different cell types: polymorphonuclear leukocytes (PMN); DMSO-differentiated HL-60 cells; murine macrophages (RAW 264.7); and TNFalpha-stimulated human endothelial cells (HUVEC). Extracellular ROS production was calculated by subtracting intracellular CL response in the presence of superoxide dismutase and catalase from the overall CL response in the absence of enzymes. CL varied considerably in dependence on the CLS and the stimulus used to evoke ROS generation. Luminol (oxidized LDL and zymosan stimulation) and isoluminol (FMLP and PMA stimulation) were the most effective CLS for PMN. Using 5 mumol/L lucigenin as CLS, small but consistent CL responses could be obtained in macrophages stimulated with PMA, zymosan or oxidized LDL. FMLP-stimulated extracellular CL in H-60 cells, HUVEC and macrophages was detected with the greatest sensitivity by pholasin. Our results demonstrate that none of the investigated CLS consistently yielded the highest CL quantum, either in different cell types with one stimulating agent or by different stimulating agents in one cell type. To get the highest CL quantum in experimental studies, we recommend optimizing the CLS depending on the cell type and the ROS-generating stimulus used. Copyright (C) 2003 John Wiley Sons, Ltd.