FLUORESCENT MICROPLATE ASSAY FOR RESPIRATORY BURST OF PMNS CHALLENGEDIN-VITRO WITH ORTHOPEDIC METALS

This report describes a simple, rapid, automated microassay for measur ing in vitro changes of oxidative burst of phagocytes following challe nge with metals for orthopedic devices. The production of reactive oxy gen species (ROS) by polymorphonuclear leukocytes (PMNs) was measured using 2',7'-dichlorofluorescin-diacetate (DCFH-DA) as fluorescent prob e. DCFH-DA enters the cells and is oxidized by ROS to fluorescent DCF. The DCF generated was directly proportional to ROS produced intracell ularly: The fluorescence intensity was read and converted to an index of ROS production by cells, in our experimental system, granulocytes ( PMNs) were isolated from normal human blood and seeded in microplates. To verify if metals could influence ROS production, chromium, cobalt, nickel, molybdenum, titanium, aluminum, and vanadium prepared as aque ous extracts in phosphate-buffered saline were tested onto PMNs using phorbolmyristate acetate (PMA) as positive control. Molybdenum, alumin um, and vanadium increased ROS generation by PMNs, while signals not d ifferent from unstimulated PMNs were recorded for chromium, cobalt, ni ckel, and titanium. The DCFH-DA microplate-based assay provides an in vitro tool for the detection of oxygen-reactive species generated by P MNs as a response to metals. (C) 1998 John Wiley & Sons, Inc.