In Vitro Glucose Depletion Assay: A Fluorescence-Based Method to Quantify Cellular Glucose Uptake via Extracellular Glucose Depletion Measurement
Transcrição
Glucose, a primary cellular energy source, enters cells via cell membrane glucose transporter proteins. Glucose gets broken down in the cell cytoplasm to pyruvate, via the glycolytic pathway, generating energy through adenosine triphosphate, or ATP, for cellular metabolism.
To measure intracellular glucose uptake, begin with an adherent culture of fibroblasts in a multi-well plate. Replace the media with glucose-free media and incubate. The cells metabolize the available glucose to produce energy, attaining a glucose-starved state.
Replace the media in one set of wells with glucose-free media containing insulin, a glucose uptake stimulator, and fluorescent deoxyglucose, a glucose analog. Treat another set of wells with glucose-free media containing fluorescent deoxyglucose.
During incubation, insulin stimulates the translocation of additional glucose transporters to the plasma membrane, facilitating increased fluorescent deoxyglucose uptake, in the absence of glucose.
Deoxyglucose is phosphorylated and not metabolized further by cells. Concurrently, the extracellular concentration of fluorescent deoxyglucose decreases.
Post-incubation, collect the media containing unutilized fluorescent deoxyglucose. Treat the cells with a lysis buffer. The detergent in the buffer forms micelles and solubilizes the cellular membranes, causing intracellular content release.
Using a microplate reader, measure the fluorescence of the cell homogenate containing intracellular fluorescent deoxyglucose and media containing unutilized extracellular fluorescent deoxyglucose.
Insulin-stimulated fibroblasts exhibit increased intracellular fluorescent deoxyglucose uptake and corresponding decreased extracellular fluorescent deoxyglucose compared to non-stimulated cells.
