A New Sensitive Chemiluminescence Probe, L-012, for Measuring the Production of Superoxide Anion by Cells

A sensitive chemiluminescence method for measuring the production of superoxide anion (O2-) by activated EoL-1 cells (human eosinophilic leukemia cell line) is described. Recently, we succeeded in synthesizing a new chemiluminescence probe, 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)dione (L-012). In the presence of L-012, activated EoL-1 cells which produce reactive oxygen species generated a marked chemiluminescence with negligible background. The L-012-dependent chemiluminescence was completely abolished by 100-300 U/ml superoxide dismutase, indicating that the main reactive oxygen species detected in this reaction was O2-. The light intensity and the sensitivity of L-012 to O2- were higher than those of other chemiluminescence probes such as luminol and Cypridina luciferin analog (MCLA). Thus, L-012 would provide an improved chemiluminescence method for measuring O2- from cells.