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

Abstract

Background: 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. Objective: We aimed to validate intraerythrocytic ROS detection by flow cytometry using DCHF-DA in healthy horses. Methods: Erythrocytes from 31 healthy horses were isolated, incubated with DCFH-DA, and either left unstimulated or stimulated with hydrogen peroxide (H 2 O 2). 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 48 hours after blood collection. The acceptable coefficient of variation (CV) was ≤20%. Results: 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 24 hours, respectively. Stimulated samples had greater fluorescence than unstimulated samples (P < .0001). Conclusions: 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.

Effects of intravenous administration of peripheral blood‐derived mesenchymal stromal cells after infusion of lipopolysaccharide in horses

“…One of the 2 whole blood samples for each horse was used for intraerythrocytic ROS measurement as previously described. 46 Briefly, samples were centrifuged at 3000 g for 5 minutes at 4°C and the erythrocyte pellet was diluted in phosphate saline buffer (PBS, pH 7.4; 137 mM NaCl [EMD Chemicals Inc, Savannah, Georgia], 10 mM Na 2 HPO 4 [Sigma‐Aldrich, St. Louis, Missouri], 2.7 mM KCl [Mallinckrodt Specialty Chemicals Co., St. Louis, Missouri], and 1.8 mM KH 2 PO 4 [Mallinckrodt Specialty Chemicals Co., St. Louis, Missouri]) supplemented with 1% w/v albumin (PBSA, Fisher Scientific, Pittsburg, Pennsylvania). The second blood sample was used for intraleukocytic ROS measurement after buffy coat separation and erythrocyte lysis.…”

“…The coefficient of variation (CV) for flow cytometric data was set at ≤20%. 46 Replicates causing the CV to be >20% were considered outliers and were removed (186 outliers out of approximately 2500 measurements).…”

“…Two samples of whole blood anticoagulated with EDTA were collected from each horse at BL, T−0.5, 0, 1, 6, 12, and 24 hours and were stored at 4°C until analysis up to 12 hours after collection. One of the 2 whole blood samples for each horse was used for intraerythrocytic ROS measurement as previously described 46 . Briefly, samples were centrifuged at 3000 g for 5 minutes at 4°C and the erythrocyte pellet was diluted in phosphate saline buffer (PBS, pH 7.4; 137 mM NaCl [EMD Chemicals Inc, Savannah, Georgia], 10 mM Na 2 HPO 4 [Sigma‐Aldrich, St. Louis, Missouri], 2.7 mM KCl [Mallinckrodt Specialty Chemicals Co., St. Louis, Missouri], and 1.8 mM KH 2 PO 4 [Mallinckrodt Specialty Chemicals Co., St. Louis, Missouri]) supplemented with 1% w/v albumin (PBSA, Fisher Scientific, Pittsburg, Pennsylvania).…”

“…Geisser‐Greenhouse's correction was used to adjust for violations of sphericity in the repeated measures analysis. The coefficient of variation (CV) for flow cytometric data was set at ≤20% 46 . Replicates causing the CV to be >20% were considered outliers and were removed (186 outliers out of approximately 2500 measurements).…”

“…One of the 2 whole blood samples for each horse was used for intraerythrocytic ROS measurement as previously described. 46 Briefly, samples were centrifuged at 3000g for…”