An in vitro protocol to study the effect of hyperglycemia on intracellular redox signaling in human retinal pigment epithelial (ARPE-19) cells.

DMEM/F12 nutrient mixture, a recommended media for ARPE-19 culture, contains glucose concentration of 17.5 mM.
But, several recent studies employed normal glucose media (5.5 mM) that was shown to affect the growth and function
of ARPE-19 cells. Here, we set a protocol to study the effect of hyperglycemia on intracellular oxidative stress and redox
status in ARPE-19 using DMEM/F12 as control. The WST-1 assay was performed to analyze the viability of ARPE-19
upon glucose treatment. The intracellular oxidative stress was measured by a dichlorofluorescein assay. The mitochondrial
membrane potential (MMP) was monitored by using a JC-10 MMP assay kit. The expression of antioxidant marker proteins
was analyzed by western blotting. Exogenous addition of glucose (7.5 and 12.5 mM) for 24 and 48 h did not change the
viability and morphology of ARPE-19 cells. Hyperglycemia increased intracellular ROS level and decreased MMP in a
dose-dependent manner. High-glucose treatment for 24 h down-regulated the protein expression of redox-specific transcription
factors Nrf-2, XBP-1 and NF-κB, and subsequently decreased the expression of HO-1, catalase, and SOD-2. This study
offers baseline information for the subsequent use of DMEM/F12 nutrient mixture to study glucose-mediated changes in
intracellular oxidative stress and redox status of ARPE-19 without affecting its basic functions.