In Vitro Glucose Depletion Assay: A Fluorescence-Based Method to Quantify Cellular Glucose Uptake via Extracellular Glucose Depletion Measurement

In This Article

Abstract

Source: Kumar, S. B. et al., Extracellular Glucose Depletion as an Indirect Measure of Glucose Uptake in Cells and Tissues Ex Vivo. J. Vis. Exp. (2022)

This video demonstrates an in vitro assay to indirectly measure intracellular glucose uptake. The assay uses a fluorescently-labeled glucose analog to establish a correlation between extracellular glucose depletion and intracellular glucose uptake.

Protocol

NOTE: All procedures must be done in a class II biosafety cabinet with the blower on and the lights off.

1. Preparation of materials

NOTE: All materials are listed in the Table of Materials.

Prepare Medium 1, centrifugation Medium 2, and storage and working solutions of fluorescent 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]-D-glucose (2-NBDG or FD-glucose) according to Table 1 in a Class II biosafety cabinet. Protect FD-glucose from light throughout the experiment by turning off all the lights under the hood.
Use r....

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Results

FD-glucose uptake graph showing insulin effect on intracellular and extracellular levels.
Figure 1: Depletion of extracellular FD-glucose correlated with the intracellular uptake in vitro. 3T3-L1 preadipocytes were incubated in a glucose-free medium for 40 min before incubation with different concentrations of FD-glucose with and without insul.......