Date of Graduation
Bachelor of Science in Biomedical Engineering
2[N-(7-nitrobenz-2-oxa-1,2-diaxol-4-yl)amino]-2-deoxyglucose (2-NBDG) a fluorescently tagged analog of FDG is ideal for in vitro studies and imaging. 2-NBDG has been proven to be an adequate marker for glucose uptake in many different types of cells . However, across the spectrum of 2-NBDG use a lack of consensus is observed for the following questions. What is the ideal time to fast cells to optimize cellular uptake of 2-NBDG? Also, what is the ideal concentration of 2-NBDG to be used when quantifying glucose uptake? Finally, what is the effect of serum on the uptake of 2-NBDG? To answer these questions and understand glucose uptake, the Balb/cfC3H 4T07 murine breast cancer cell line was fasted at varying time points between 0 and 150 minutes. Cell viability was evaluated for these time points using Promega’s (Madison, WI) CellTiter-Glo® luminescent assay. Cells were also plated into 35mm glass bottom dishes, incubated for 24 hours, and fasted for varying times between 0 and 150 minutes. 400µM of 2-NBDG was introduced for 20 minutes and uptake was quantified using fluorescence microscopy. The peak of cell viability and glucose uptake was compared to find the optimal fasting time. Once fasting studies were complete, cells were fasted according to ideal conditions and concentration dependency of 2-NBDG was investigated. It was found that 4T07 cell viability is significantly decreased by 60 minutes of fasting cells in DMEM (-) glucose in the absence of 10% serum. The addition of 10% serum to the DMEM (-) glucose prolongs the fasting range to at least 150 minutes. 2-NBDG uptake is higher with the addition of 10% serum to DMEM (-) glucose in 20 minute fasting conditions. Also, 400µM 2-NBDG is the ideal concentration to optimize cell viability, cost effectiveness, and uptake.
Folgmann, Drew, "Optimized Protocol for Measuring 2-NBDG Uptake as a Cellular Marker of Glycolytic Demand" (2016). Biomedical Engineering Undergraduate Honors Theses. 31.