An in vitro protocol to study the effect of hyperglycemia on intracellular redox signaling in human retinal pigment epithelial (ARPE-19) cells.
IR@CFTRI: CSIR-Central Food Technological Research Institute, Mysore
View Archive Info| Field | Value | |
| Relation |
http://ir.cftri.com/13954/
https://doi.org/10.1007/s11033-019-04597-x |
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| Title |
An in vitro protocol to study the effect of hyperglycemia
on intracellular redox signaling in human retinal pigment epithelial
(ARPE-19) cells.
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| Creator |
Arpitha, H. S.
Sowya Shree, G. Ganesan, P. |
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| Subject |
04 Diabetes Mellitus
32 Antioxidants |
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| Description |
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.
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| Date |
2019
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| Type |
Article
PeerReviewed |
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| Format |
pdf
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| Language |
en
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| Identifier |
http://ir.cftri.com/13954/1/Molecular%20Biology%20Reports%20%282019%29%20461263%E2%80%931274.pdf
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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