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

Published: April 30, 2023

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…

Representative 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 insulin (1.7 mM; 40 min incubation). (A, B) Dose-dependent uptake of FD-glucose (A) and extracel…

開示

The authors have nothing to disclose.

Materials

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

タグ

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記事を引用
In Vitro Glucose Depletion Assay: A Fluorescence-Based Method to Quantify Cellular Glucose Uptake via Extracellular Glucose Depletion Measurement. J. Vis. Exp. (Pending Publication), e21212, doi: (2023).

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