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

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.

1. 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.
2. Use r....

Results

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
3T3-L1 mouse fibroblasts	ATCC	CL-173	Cell line
96-well plates	Falcon	353227	Plastic ware
BioTek Synergy H1 modular multimode microplate reader (Fisher Scientific, US)	Fisher Scientific, US	 B-SHT	Device
Bovine serum	Gibco/ThermoFisher	161790-060	Cell culture
Calf serum	Gibco/ThermoFisher	26010-066	Cell culture
Cell incubator	Forma	Series II Water Jacket	Device
Dimethylsulfoxide (DMSO)	Sigma LifeScience	D2650-100mL	Reagent
Dulbecco's Modified Eagle Medium	Gibco/ThermoFisher	 11965-092	Cell culture
Ethanol	Sigma Aldrich	E7023-500mL	Reagent
Fluorescent 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]-D-glucose)	Sigma	72987-1MG	Assay
Glucose-free and phenol red-free DMEM	Gibco/ThermoFisher	A14430-01	Cell culture
Human insulin 10 mg/mL	MilliporeSigma	Cat N 91077C	Reagent
Penicillin/streptomycin (P/S)	Gibco/ThermoFisher	15140-122	Cell culture
Phosphate buffered solution	Sigma-Aldrich	DA537-500 mL	Cell culture
Radioimmunoprecipitation assay lysis buffer	Santa Cruz Biotechnology	sc-24948	Assay
Trypsin-EDTA (0.05%)	Gibco/ThermoFisher	 25300-054	Cell culture