Abstract
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.
Highlights
The scientific research on reactive oxygen species (ROS), for a deeper insight into their biological functions and/or deleterious effects, still is a matter of intense research
Despite the usefulness of the D-optimal experimental design, this method is not usually applied to biologic matrices, being used here, for the first time, to optimize the experimental conditions for the in vitro detection of ROS produced by human blood cells, from healthy donors, following stimulation by a Oxidative Medicine and Cellular Longevity potent inflammatory mediator, phorbol-12-myristate13-acetate (PMA), using different fluorescent probes, 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
Taking into account that all tested dilutions of human blood originated a good range of oxidation percentage of the probe, we choose the 1 : 20 dilution in order to use less quantity of human blood in each assay, making the biologic sample more profitable
Summary
The scientific research on reactive oxygen species (ROS), for a deeper insight into their biological functions and/or deleterious effects, still is a matter of intense research. Fluorescent probes have been mainly used to detect ROS in isolated cells, namely neutrophils [1, 2]. The isolation process itself often leads to artifactual cell activation, which represents an experimental confounder, being expensive and time-consuming [3]. Human blood is the most complex biological matrix that better resembles the physiological environment. There are just a few reports in literature about the detection of reactive species in human blood [3,4,5], but none of them described the experimental optimization of the method
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