Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba; Centro de Investigaciones en Bioquímica Clínica e Inmunología (CIBICI), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET).
Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba; Instituto de Investigación Médica Mercedes y Martín Ferreyra (INIMEC), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET).
J Vis Exp. 2022 May 13(183). doi: 10.3791/63337.
The redox balance has an important role in maintaining cellular homeostasis. The increased generation of reactive oxygen species (ROS) promotes the modification of proteins, lipids, and DNA, which finally may lead to alteration in cellular function and cell death. Therefore, it is beneficial for cells to increase their antioxidant defense in response to detrimental insults, either by activating an antioxidant pathway like Keap1/Nrf2 or by improving redox scavengers (vitamins A, C, and E, β-carotene, and polyphenols, among others). Inflammation and oxidative stress are involved in the pathogenesis and progression of retinopathies, such as diabetic retinopathy (DR) and retinopathy of prematurity (ROP). Since Müller glial cells (MGCs) play a key role in the homeostasis of neural retinal tissue, they are considered an ideal model to study these cellular protective mechanisms. In this sense, quantifying ROS levels with a reproducible and simple method is essential to assess the contribution of pathways or molecules that participate in the antioxidant cell defense mechanism. In this article, we provide a complete description of the procedures required for the measurement of ROS with DCFH-DA probe and flow cytometry in MGCs. Key steps for flow cytometry data processing with the software are provided here, so the readers will be able to measure ROS levels (geometric means of FITC) and analyze fluorescence histograms. These tools are highly helpful to evaluate not only the increase in ROS after a cellular insult but also to study the antioxidant effect of certain molecules that can provide a protective effect on the cells.
氧化还原平衡在维持细胞内稳态方面起着重要作用。活性氧（ROS）的产生增加会促进蛋白质、脂质和 DNA 的修饰，最终可能导致细胞功能的改变和细胞死亡。因此，细胞通过激活 Keap1/Nrf2 等抗氧化途径或增加氧化还原清除剂（维生素 A、C、E、β-胡萝卜素和多酚等）来增加其抗氧化防御能力，从而抵御有害刺激是有益的。炎症和氧化应激参与了糖尿病性视网膜病变（DR）和早产儿视网膜病变（ROP）等视网膜病变的发病机制和进展。由于 Müller 胶质细胞（MGC）在神经视网膜组织的内稳态中起着关键作用，因此它们被认为是研究这些细胞保护机制的理想模型。从这个意义上说，用一种可重复且简单的方法来定量 ROS 水平对于评估参与抗氧化细胞防御机制的途径或分子的贡献至关重要。在本文中，我们提供了使用 DCFH-DA 探针和流式细胞术测量 MGC 中 ROS 的完整描述。本文还提供了用于处理流式细胞术数据的软件的关键步骤，以便读者能够测量 ROS 水平（FITC 的几何平均值）并分析荧光直方图。这些工具不仅有助于评估细胞受到刺激后 ROS 的增加，还可以研究某些分子的抗氧化作用，这些分子可以为细胞提供保护作用。
