Quantification of reactive oxygen species generated by alveolar macrophages using lucigenin-enhanced chemiluminescence--methodical aspects.

Alveolar macrophages lavaged from bovine lungs using Ca2(+)- and Mg2(+)-deprived saline containing EGTA for calcium chelation were cultivated in RPMI-1640 medium. The generation of reactive oxygen species (ROS) was assessed by determination of lucigenin-enhanced chemiluminescence (LUC-CL) using a LB 9505 C Biolumat. Monitoring of LUC-CL depends on various methodical parameters: besides medium constituents, the method of cell harvesting and the time-schedule of the protocol appear to be essential parameters that influence ROS-generation. In addition, the cell number (cell density) as well as the ratio of cell number to particle mass influence the amount of ROS-generation. Following exposure of cells to micronized quartz, we observed a dose-related increase in the generation of ROS.