Measurement of Extracellular Fluorescent Glucose Analog Depletion: A Surrogate Measurement Technique to Assess Glucose Uptake in Mouse Organs Ex Vivo
Transcript
In tissues, extracellular glucose enters cells via facilitated diffusion through specific glucose transporters.
Glucose gets broken down to pyruvate in the cell cytoplasm via glycolysis, a metabolic pathway, generating energy in the form of adenosine triphosphate, or ATP, for cellular metabolic processes.
To measure glucose uptake ex vivo, begin with freshly harvested small-sized mouse liver tissue. Add suitable buffer to maintain physiological conditions.
Next, transfer the tissue to a multi-well plate containing glucose-free media. Follow it by incubation in glucose-free media supplemented with deoxyglucose, a fluorescently-labeled glucose analog. The small size of the tissue allows the entry of deoxyglucose.
Deoxyglucose enters the cells through glucose transporters, in the absence of glucose. Within the cells, deoxyglucose undergoes phosphorylation but is not metabolized further, leading to its accumulation.
Collect the media containing unutilized fluorescently-labeled deoxyglucose at desired time points. Using a microplate reader, measure the fluorescence of the extracellular deoxyglucose, which can be correlated with the intracellular tissue uptake of deoxyglucose.
