Quantification of Reactive Oxygen Species Using 2′,7′-Dichlorofluorescein Diacetate Probe and Flow-Cytometry in Müller Glial Cells

作者： María V. Vaglienti1,2, Paula V. Subirada1,3, Pablo F. Barcelona1,2, Gustavo Bonacci1,2, María C. Sanchez1,2 1Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas,Universidad Nacional de Córdoba, 2Centro de Investigaciones en Bioquímica Clínica e Inmunología (CIBICI),Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), 3Instituto de Investigación Médica Mercedes y Martín Ferreyra (INIMEC),Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)

简介：

Overview

This study presents a protocol for detecting cellular reactive oxygen species (ROS) levels using the 2′,7′-dichlorofluorescein diacetate (DCFH-DA) probe in Müller glial cells (MGCs). The method is designed to be accessible, reproducible, and quantifies ROS using flow cytometry.

Key Study Components

Area of Science

• Cellular Biology
• Oxidative Stress
• Neuroscience

Background

• Reactive oxygen species (ROS) play a critical role in various cellular processes.
• Measurement of ROS is essential for understanding oxidative stress in cells.
• The DCFH-DA probe is a widely used method for detecting ROS.
• The protocol emphasizes reproducibility and ease of use.

Purpose of Study

• To provide a clear protocol for detecting ROS in Müller glial cells.
• To enable quantification of oxidative stress in neuronal cells.
• To facilitate research on the role of ROS in glial function and neurobiology.

Methods Used

• Cell culture of Müller glial cells was performed to create the biological model.
• Cells were treated with antioxidants and ROS inducers to assess ROS levels.
• The DCFH-DA probe was used for ROS detection and quantified using flow cytometry.
• Critical steps included washing cells with PBS and controlling light exposure during assays.

Main Results

• The protocol effectively measured cellular ROS levels in Müller glial cells.
• Flow cytometry provided a quantitative analysis of ROS accumulation.
• Results confirmed the feasibility of using the DCFH-DA probe under specified conditions.

Conclusions

• This study establishes a reliable method for assessing oxidative stress in glial cells.
• The protocol simplifies ROS measurement, enhancing accessibility for researchers.
• Findings have implications for understanding oxidative stress in neurobiology and potential therapeutic targets.

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