Validation of a flow cytometric assay to detect intraerythrocytic reactive oxygen species in horses

Abstract

Oxidative stress refers to the accumulation of reactive oxygen species (ROS). Most assays for ROS detection are costly, laborious, and usually use indirect markers. The use of 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) is a possible alternative. This substance becomes a fluorochrome when oxidized by ROS, with the resultant fluorescence proportional to ROS concentration. Erythrocytes are highly exposed to ROS, resulting in cell damage and consequently impaired oxygen delivery. The effects of this exposure in physiologic and pathologic conditions necessitate an improvement in ROS detection methods. We aimed to validate intraerythrocytic ROS detection by flow cytometry using DCHF-DA in healthy horses. Erythrocytes from 31 healthy horses were isolated, incubated with DCFH-DA, and either left unstimulated or stimulated with hydrogen peroxide (H2 O2 ). For specificity, each cellular component of blood was separated and plotted according to its size and complexity. Samples were run in triplicate for intra-assay precision and five consecutive times for inter-assay repeatability. Stability was determined by analyzing the same sample up to 48hours after blood collection. The acceptable coefficient of variation (CV) was ≤20%. The intra-assay CV was 1.7% and 13.3%, and the inter-assay CV was 4.8% and 17.8% for unstimulated and stimulated samples, respectively. Unstimulated and stimulated samples were stable for up to 48 and 24hours, respectively. Stimulated samples had greater fluorescence than unstimulated samples (P<.0001). This flow cytometric assay demonstrated adequate specificity, precision, and stability and is, therefore, a promising technique with multiple applications for studying oxidative stress in horses.