Fluorescent microplate assay for respiratory burst of PMNs challengedin vitro with orthopedic metals

Abstract

This report describes a simple, rapid, automated microassay for measuring in vitro changes of oxidative burst of phagocytes following challenge with metals for orthopedic devices. The production of reactive oxygen species (ROS) by polymorphonuclear leukocytes (PMNs) was measured using 2',7'-dichlorofluorescin-diacetate (DCFH-DA) as fluorescent probe. DCFH-DA enters the cells and is oxidized by ROS to fluorescent DCF. The DCF generated was directly proportional to ROS produced intracellularly: 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 aqueous extracts in phosphate-buffered saline were tested onto PMNs using phorbolmyristate acetate (PMA) as positive control. Molybdenum, aluminum, and vanadium increased ROS generation by PMNs, while signals not different from unstimulated PMNs were recorded for chromium, cobalt, nickel, and titanium. The DCFH-DA microplate-based assay provides an in vitro tool for the detection of oxygen-reactive species generated by PMNs as a response to metals.

Optical probes for detection and quantification of neutrophils’ oxidative burst. A review

“…This conclusion was also drawn by Walrand et al [29], after using a NOS inhibitor in PMA-activated neutrophils with a resulting decrease of DCFH fluorescence. Ciappetti et al [51], described a microassay based on DCFH-DA method, for measuring in vitro changes of oxidative burst of neutrophils induced by several metals. The authors' referred that this methodology allows the use of a small blood volume, is technically simple and provide quantitative results.…”

Cytotoxic effect of bone cements in HL-60 cells: Distinction between apoptosis and necrosis

“…Production of ROS by HL60 cells was quantitated by the 2Ј,7Ј-dichlorodihydrofluorescein diacetate method, 15 based on the oxygen-dependent oxidation of DCFH-DA to DCF.…”

The potential role of cobalt ions released from metal prosthesis on the inhibition of Hv1 proton channels and the decrease in Staphyloccocus epidermidis killing by human neutrophils

“…In contrast, Co 2þ impaired the ability of neutrophils to produce ROS in response to PMA or to zymosan. These findings are consistent with two earlier studies reporting that Co 2þ does not promote ROS production in human neutrophils [63,64] but instead inhibits the O 2 À production evoked by the chemotactic peptide f-MLP [ [63,64]. Thus, in neutrophils, Co 2þ does not generate ROS but instead inhibits the production of ROS by the phagocytic NADPH oxidase.…”