Department of Comparative Pathobiology, Purdue University College of Veterinary Medicine, West Lafayette, Indiana, USA.
Department of Veterinary Clinical Sciences, Purdue University College of Veterinary Medicine, West Lafayette, Indiana, USA.
Vet Clin Pathol. 2021 Mar;50(1):20-27. doi: 10.1111/vcp.12976. Epub 2021 Mar 1.
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 (H O ). 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%.
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).
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.
氧化应激是指活性氧（ROS）的积累。大多数 ROS 检测方法既昂贵又费力，通常使用间接标志物。使用 2'，7'-二氯二氢荧光素二乙酸酯（DCFH-DA）可能是一种替代方法。这种物质被 ROS 氧化后成为荧光染料，其荧光强度与 ROS 浓度成正比。红细胞极易受到 ROS 的影响，导致细胞损伤，进而影响氧气输送。在生理和病理条件下，这种暴露的影响需要改进 ROS 检测方法。
我们旨在使用 DCFH-DA 通过流式细胞术验证健康马的红细胞内 ROS 检测。
从 31 匹健康马中分离红细胞，用 DCFH-DA 孵育，然后不刺激或用过氧化氢（H2O2）刺激。为了特异性，血液的每个细胞成分都根据其大小和复杂性进行分离和绘制。每个样本进行三次重复以评估内实验精度，连续五次重复以评估间实验重复性。稳定性通过分析采血后长达 48 小时的同一样本来确定。可接受的变异系数（CV）应≤20%。
未刺激和刺激样本的内实验 CV 分别为 1.7%和 13.3%，间实验 CV 分别为 4.8%和 17.8%。未刺激和刺激样本在长达 48 小时和 24 小时内均稳定。刺激样本的荧光强度大于未刺激样本（P<0.0001）。
该流式细胞术检测方法具有足够的特异性、精度和稳定性，因此是一种很有前途的技术，可用于研究马的氧化应激。
