Am J Vet Res. 2021 May;82(5):343-351. doi: 10.2460/ajvr.82.5.343.
To validate the use of a flow cytometric assay that uses 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) to measure reactive oxygen species in the erythrocytes of healthy dogs.
50 healthy adult dogs.
Erythrocytes were incubated with DCFH-DA or a vehicle control (dimethyl sulfoxide), then incubated with (stimulated) or without (unstimulated) hydrogen peroxide. The flow cytometric assay was evaluated for specificity with increasing concentrations of DCFH-DA and hydrogen peroxide, and a polynomial regression line was applied to determine optimal concentrations. For precision, samples were analyzed 5 consecutive times for determination of intra- and interassay variability. Stability of samples stored at 4°C for up to 48 hours after blood collection was determined with flow cytometric analysis. Coefficient of variation (CV) was considered acceptable at 20%. Baseline measurements were used to determine an expected range of median fluorescence intensity for unstimulated erythrocytes incubated with DCFH-DA.
Erythrocytes were successfully isolated, and stimulated samples demonstrated higher median fluorescence intensity, compared with unstimulated samples. The intra-assay CV was 11.9% and 8.9% and interassay CV was 11.9% and 9.1% for unstimulated and stimulated samples, respectively. Unstimulated samples were stable for up to 24 hours, whereas stimulated samples were stable for up to 48 hours.
Flow cytometry for the measurement of reactive oxygen species in the erythrocytes of healthy dogs by use of DCFH-DA had acceptable specificity, precision, and stability. Flow cytometry is a promising technique for evaluating intraerythrocytic oxidative stress for healthy dogs.
验证使用 2'，7'-二氯二氢荧光素二乙酸酯（DCFH-DA）测量健康犬红细胞中活性氧的流式细胞术检测方法的有效性。
50 只健康成年犬。
将红细胞与 DCFH-DA 或载体对照（二甲基亚砜）孵育，然后孵育（刺激）或不孵育（未刺激）过氧化氢。使用递增浓度的 DCFH-DA 和过氧化氢评估流式细胞术检测方法的特异性，并应用多项式回归线确定最佳浓度。为了精密度，连续 5 次分析样品以确定批内和批间变异性。采集血液后 48 小时内，通过流式细胞分析确定在 4°C 储存的样品稳定性。变异系数（CV）可接受范围为 20%。使用基线测量值确定未刺激的用 DCFH-DA 孵育的红细胞的中位荧光强度的预期范围。
成功分离红细胞，与未刺激的样本相比，刺激的样本显示出更高的中位荧光强度。未刺激样本的批内 CV 为 11.9%和 8.9%，刺激样本的批内 CV 为 11.9%和 9.1%。未刺激样本在 24 小时内稳定，而刺激样本在 48 小时内稳定。
使用 DCFH-DA 通过流式细胞术测量健康犬红细胞中的活性氧具有可接受的特异性、精密度和稳定性。流式细胞术是评估健康犬红细胞内氧化应激的有前途的技术。
