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Updated: May 28, 2026
Quantification of Reactive Oxygen Species Using 2′,7′-Dichlorofluorescein Diacetate Probe and Flow-Cytometry in Müller Glial Cells
Published on: May 13, 2022
Rapamycin reduces reactive oxygen species in cultured human corneal endothelial cells
Young Joo Shin1, Doo Young Cho, Tae Young Chung
1Department of Ophthalmology, Hallym University College of Medicine, Seoul, Republic of Korea. schinn@hanmail.net
Purpose:
To investigate the protective effect of rapamycin on oxidative stress-induced cell death of human corneal endothelial cells (HCECs).
Methods:
HCECs were cultured according to previously published methods. With treatment of 0 mM or 5 mM of tert-butyl hydroperoxide (tBHP) with various concentrations (0, 25 and 50 nM) of rapamycin, reactive oxygen species (ROS) production was measured using an oxidation-sensitive fluorescent probe, 2'7'-dichlorofluorescin diacetate (DCFH-DA, USA) methods. Cell viability was assayed by the method of Cell Counting Kit-8 (CCK-8, Wako). The levels of cellular glutathione were also assessed enzymatically with glutathione reductase by using a commercial glutathione (GSH) assay kit (Cayman Chemical, USA).
Results:
Rapamycin reduced 2'7'-dihydrodichlorofluorescein oxidation and increased GSH in HCECs. Rapamycin significantly inhibited tBHP-induced ROS production. Cells treated with rapamycin showed higher viability compared to control at 5 mM tBHP. Rapamycin effectively protected HCECs from ROS-induced cell death through increasing intracellular GSH.
Conclusion:
Our data suggest that rapamycin protects HCECs from oxidative injury-mediated cell death via inhibition of ROS production and enhancement of GSH.
