Spatially Resolved Measurement of Dynamic Glucose Uptake in Live Ex Vivo Tissues
Dunn, Austin, Chemistry - Graduate School of Arts and Sciences, University of Virginia
Pompano, Rebecca, AS-Chemistry, University of Virginia
Highly proliferative cells depend heavily on glycolysis as a source of energy and biological precursor molecules, and glucose uptake is a useful readout of this aspect of metabolic activity. Glucose uptake is commonly quantified by using flow cytometry for cell cultures and positron emission tomography for organs in vivo. However, methods to detect spatiotemporally resolved glucose uptake in intact tissues are far more limited, particularly those that can quantify changes in uptake over time in specific tissue regions and cell types. Using lymph node metabolism as a case study, we developed a novel assay of dynamic and spatially resolved glucose uptake in living tissue by combining ex vivo tissue slice culture with a fluorescent glucose analogue. Live slices of murine lymph node were treated with the glucose analogue 2-[N-(7-nitrobenz-2-oxa-1,3-dia-xol-4-yl)amino]-2-deoxyglucose (2-NBDG). Incubation parameters were optimized to differentiate glucose uptake in activated versus naive lymphocytes. Confocal microscopy of treated tissues confirmed that the 2-NBDG signal was intracellular. The assay was readily multiplexed with live immunofluorescence labelling to generate maps of 2-NBDG uptake across tissue regions, revealing highest uptake in T cell-dense regions. Uptake was predominantly localized to lymphocytes rather than stromal cells. Because 2-NBDG washed out of the tissue over time, the assay was repeatable in the same slices before and after T cell activation to reveal which tissue regions were most responsive to stimulation. We anticipate that this assay will serve as a broadly applicable, user-friendly platform to quantify dynamic metabolic activities in complex tissue microenvironments.
MS (Master of Science)