Comparative detection method of early onset cytokine-induced stress in β-cells (INS-1E)
| Autor: | Natasha Beukes, Carminita L. Frost, R.-A. Levendal, Earl Prinsloo |
|---|---|
| Rok vydání: | 2018 |
| Předmět: |
Cell Survival
medicine.medical_treatment Biomedical Engineering Sulforhodamine B Bioengineering Endoplasmic Reticulum Applied Microbiology and Biotechnology Proinflammatory cytokine Cell Line chemistry.chemical_compound Downregulation and upregulation Insulin-Secreting Cells Drug Discovery medicine Animals Viability assay Cell Proliferation Chemistry Process Chemistry and Technology Endoplasmic reticulum Insulin General Medicine Endoplasmic Reticulum Stress Cell biology Rats Cytokine Unfolded protein response Molecular Medicine Cytokines Biotechnology |
| Zdroj: | Biotechnology and applied biochemistry. 66(3) |
| ISSN: | 1470-8744 |
| Popis: | β-Cells contain a prominent endoplasmic reticulum (ER), disrupting ER homeostasis and function, activating the unfolded protein response (UPR). Currently, no direct protocols measure the UPR initiation. Current methods to measure ER stress include the quantification of nitric oxide (NO) (indirect method), Western blotting, and qRT-PCR of downstream components. However, these methods do not account for the overlap with mitochondrial dysfunction. In this study, INS-1E cells were exposed to proinflammatory cytokines to induce ER stress, as determined using NO, thioflavin T (ThT) binding, and β-cell functionality (insulin production). ER stress was confirmed through the upregulation of CHOP. Cell viability was monitored using MTT, sulforhodamine B, and the xCELLigence system. Morphological changes were monitored using electron microscopy. IL-1β exposure-induced β-cell stress after 4 H, decreased insulin levels, and increased thioflavin binding were noted. Increased NO production was only detected after 10 H, highlighting its lack of sensitivity, and the need for a continuous, selective, rapid, convenient, and economical detection method for early onset of ER stress. Standard methods (MTT and NO) failed to detect early ER stress. The xCELLigence coupled with a functional assay such as the detection of insulin levels or ThT are better predictors of ER stress in INS-1E cells. |
| Databáze: | OpenAIRE |
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