Date of Graduation
5-2021
Document Type
Thesis
Degree Name
Bachelor of Science in Biomedical Engineering
Degree Level
Undergraduate
Department
Biomedical Engineering
Advisor/Mentor
Quinn, Kyle P.
Abstract
Hydroxyl radicals, superoxides, and hydrogen peroxides are common reactive oxygen species (ROS) that are produced by the mitochondria during cellular respiration [1]. Signal transduction pathways in the mitochondria are used to maintain the balance of ROS. However, when these mechanisms fail to regulate ROS properly, it causes oxidative damage and can lead to the development of various diseases such as cancer, respiratory, neurodegenerative, and cardiovascular diseases. L-buthionine sulfoximine (BSO) has been used previously to induce oxidative stress by inhibiting the γ-GCS enzyme used to synthesize glutathione, an antioxidant used to convert ROS to water [3]. BSO reduces glutathione production, thus increasing the concentration of superoxides. The objective of this study is to explore a method to quantify the concentration of superoxide in NIH 3T3 fibroblast cells treated with BSO. MitoSOXTM Red Mitochondrial Superoxide Indicator, a novel fluoroprobe used to detect superoxide found in the mitochondria of live cells, was used in this study to quantify the concentration of ROS and NucBlue was used to label the nucleus. An Olympus Fluoview FV10i Laser Scanning Confocal Microscope was used to detect the fluorescence of MitoSOX and NucBlue. The 405 nm and 473 nm lasers were used to excite the fluorescence of NucBlue and MitoSOX, respectively. A 461 nm emission filter and a 527 nm emission filter were used to capture the fluorescence of NucBlue and MitoSOX fluorophores, respectively. Overall, this method successfully quantified the concentration of superoxides present and showed that increasing BSO causes cellular retraction and apoptosis. BSO also increased the concentration of superoxides in a step like manner. The step like increase in superoxide could be caused by how potent BSO is in small concentrations. At higher concentrations, BSO caused a clear decrease in the viability of the cells.
Keywords
BSO, Superoxide, Oxidative stress, MitoSOX
Citation
Townsley, R. (2021). Quantification of Superoxide Production In Cells Treated with L-buthionine Sulfoximine. Biomedical Engineering Undergraduate Honors Theses Retrieved from https://scholarworks.uark.edu/bmeguht/104