How much iron is good? Revisiting the role of ferrous iron supplementation in cultured cells using genetically encoded NO biosensors

Sevimli, Gülşah (2021) How much iron is good? Revisiting the role of ferrous iron supplementation in cultured cells using genetically encoded NO biosensors. [Thesis]

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Although ferrous iron (Fe2+) is an essential cofactor for many metalloproteins, most cell culture media lack or only contain insufficient amounts of this transient metal, resulting in iron (II) deficiency in cultured cells. Well-established techniques permit quantification of Fe2+ in cells with high accuracy; however, these techniques do not provide information about the iron-dependent functionality of metalloprotein. In this study, we demonstrated that genetically encoded nitric oxide probe (geNOps), which comprises a non-heme ironbinding protein (GAF) fused with a fluorescent protein, can be utilized as an indirect model system to study the iron-dependency in cultured cells. In the presence of NO, the GAF domain can be reversibly nitrosylated causing changes in the fluorescence intensity in a concentration-dependent manner. We showed that long-term supplementation of Fe2+ in cultured cells has no effect on the functionality of geNOps. Only short-term provision of freshly prepared Fe2+ combined with vitamin C activated the metalloprotein. The distribution of Fe2+ in cells after short-term treatment was observed with the FeRhoNox1 and the accumulation of total iron was determined using high-resolution light microscopy techniques and Perls/DAB staining. Employing the design of experiment approaches, we optimized the Fe2+ loading procedure regarding Fe2+ and vitamin C concentrations and the duration of cell treatment. This approach yielded a non-toxic Fe2+ supplementation procedure that is essential for cell integrity and the full functionality of metalloprotein. In conclusion, this study exploited a metalloprotein readout to extrapolate the Fe2+ uptake capability of cells under cell culture conditions.
Item Type: Thesis
Uncontrolled Keywords: cell culture media. -- ferrous iron (Fe2+). -- genetically encoded NO probes (geNOps). -- vitamin C. -- ferröz demir (Fe2+). -- genetik kodlanmış NO probları (geNOps). -- hücre kültürü besiyeri.
Subjects: T Technology > TA Engineering (General). Civil engineering (General) > TA164 Bioengineering
Divisions: Faculty of Engineering and Natural Sciences > Academic programs > Biological Sciences & Bio Eng.
Faculty of Engineering and Natural Sciences
Depositing User: Dila Günay
Date Deposited: 05 Jul 2022 14:34
Last Modified: 26 Jul 2022 11:09

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