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

Arpitha, H. S. and Sowya Shree, G. and Ganesan, P. (2019) An in vitro protocol to study the effect of hyperglycemia on intracellular redox signaling in human retinal pigment epithelial (ARPE-19) cells. Molecular Biology Reports, 46. pp. 1263-1274. ISSN 0301-4851

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Abstract

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.

Item Type:	Article
Uncontrolled Keywords:	Glucose · ARPE-19 · Oxidative stress · Redox transcription factors · Antioxidant enzymes
Subjects:	600 Technology > 01 Medical sciences > 04 Diabetes Mellitus 600 Technology > 08 Food technology > 32 Antioxidants
Divisions:	Molecular Nutrition
Depositing User:	Food Sci. & Technol. Information Services
Date Deposited:	20 Mar 2019 08:43
Last Modified:	20 Mar 2019 08:43
URI:	http://ir.cftri.res.in/id/eprint/13954

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