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Oxidative Medicine and Cellular Longevity
Volume 2019, Article ID 7198484, 11 pages
Research Article

Optimization of Experimental Settings for the Assessment of Reactive Oxygen Species Production by Human Blood

LAQV, REQUIMTE, Laboratory of Applied Chemistry, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, 4050-313 Porto, Portugal

Correspondence should be addressed to Eduarda Fernandes; tp.pu.ff@sacarge and Marisa Freitas; tp.pu.ff@satierfasiram

Received 20 June 2018; Revised 27 September 2018; Accepted 21 October 2018; Published 10 January 2019

Guest Editor: Ayman M. Mahmoud

Copyright © 2019 Tânia Soares et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


The purpose of an experimental design is to improve the productivity of experimentation. It is an efficient procedure for planning experiments, so the data obtained can be analyzed to yield a valid and objective conclusion. This approach has been used as an important tool in the optimization of different analytical approaches. A D-optimal experimental design was used here, for the first time, to optimize the experimental conditions for the detection of reactive oxygen species (ROS) produced by human blood from healthy donors, a biological matrix that better resembles the physiologic environment, following stimulation by a potent inflammatory mediator, phorbol-12-myristate-13-acetate (PMA). For that purpose, different fluorescent probes were used, as 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), 2-[6-(4-amino)-phenoxy-3H-xanthen-3-on-9-yl] benzoic acid (APF), and 10-acetyl-3,7-dihydroxyphenoxazine (amplex red). The variables tested were the human blood dilution, and the fluorescent probe and PMA concentrations. The experiments were evaluated using the Response Surface Methodology and the method was validated using specific compounds. This model allowed the search for optimal conditions for a set of responses simultaneously, enabling, from a small number of experiments, the evaluation of the interaction between the variables under study. Moreover, a cellular model was implemented and optimized to detect the production of ROS using a yet nonexplored matrix, which is human blood.