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

Gabriela Ciapetti, Donatella Granchi, Elisabetta Verri, Lucia Savarino, Elisabetta Cenni, Franca Savioli, Arturo Pizzoferrato

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Pages (from-to)455-460
Number of pages6
JournalJournal of Biomedical Materials Research
Volume41
Issue number3
DOIs
Publication statusPublished - Sep 5 1998

Keywords

  • 2',7'- dichlorofluorescin-diacetate
  • Granulocytes (PMNs)
  • Metals
  • Reactive oxygen species

ASJC Scopus subject areas

  • Biomedical Engineering
  • Biomaterials

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