Original ContributionSystematic study on ROS production induced by oleic, linoleic, and γ-linolenic acids in human and rat neutrophils
Section snippets
Material and methods
RPMI 1640 medium, Hepes, penicillin, and streptomycin were purchased from Invitrogen (Carlsbad, CA, USA). Fatty acids, glutamine, luminol, lucigenin, cytochrome c, hydroethidine, peroxidase type II, hydrogen peroxide, phenol red, Histopaque, oyster glycogen, and trypan blue were supplied by Sigma Chemical Co (St. Louis, MO, USA). Fatty acids were dissolved in ethanol. The final concentration of ethanol in the assay medium did not exceed 0.05%. A preliminary experiment showed that ethanol at
Results
Appropriate controls were carried out using 10, 25, 50, 100, and 200 μM oleic, linoleic, and γ-linolenic acids in the assays (luminol, lucigenin, and phenol red) without cells. The three fatty acids did not directly affect the luminol, lucigenin, and phenol red assays. To test for possible interference by the fatty acids in the reaction of ROS with the reagents, peroxide was added to the phenol red assay, and xanthine and xanthine oxidase were added to the lucigenin and luminol assays, without
Discussion
ROS production by neutrophils is primarily associated with phagocyte defense against foreign organisms and occurs mainly through the NADPH oxidase complex. NADPH oxidase is assembled and activated either in the plasma membrane or in the membrane of internalized phagosomes. The reactive oxygen species generated will then either be released from the cells (activation in the plasma membrane) or be retained inside the phagocyte (activation in the phagosomal membrane) [1], [2], [28], [29].
Acknowledgments
The authors are indebted to the Fundação de Amparo à Pesquisa do Estado de São Paulo and to the Conselho Nacional de Desenvolvimento Científico e Tecnológico for financial support.
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