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.
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.
2. 3T3-L1 cell culture and maintenance
3. Starvation of 3T3-L1 cells
4. Preparation of FD-glucose solutions with different concentrations
NOTE: The experiment in Figure 1 examines extracellular and intercellular media with and without stimulation with insulin.
5. Treating starved 3T3-L1 cells
6. Extracellular and intercellular measurements for stimulated 3T3-L1 cells
Table 1: Preparation of culture media and FD-glucose solutions.
Solution/Medium | Components |
Ethanol:DMSO (1:1/v/v) | Ethanol (200 µL) and DMSO (200 µL) in 1-1.5 mL tube; use cell culture grade ethanol and DMSO |
Medium 1 | DMEM (89 mL), Penicillin/streptomycin (1%) (1 mL) and Calf serum (10%) (10 mL) in sterile 50 mL tube |
Centrifugation Medium 2 | DMEM (89 mL), Penicillin/streptomycin (1%) (1 mL) and Bovine serum (10%) (10 mL) in sterile 50 mL tube |
Storage FD-glucose solution 5 mg/mL (14.5 mM) | FD glucose (1 mg) and Ethanol:DMSO (1:1/v/v) (200 µL) in 0.5 mL tube; store at -80 °C, preferentially under argon or nitrogen atmosphere |
Working FD-glucose solution 5 µg/mL (14.5 mM) | Storage FD glucose solution 5 mg/mL (1 µL), Glucose free DMEM (999 µL); prepare immediately before experiment. |
Table 2: Preparation of FD-glucose solutions with different concentrations for experiments shown in Figure 1.
Dilution | Control | 1:2×103 | 1:5×103 | 1:1×104 | 1:2.5×104 | 1:5×104 | 1:1×105 |
Concentration (µg/mL) | 0 | 2.5 | 1 | 0.5 | 0.2 | 0.1 | 0.05 |
Glucose-free DMEM (µL) | 1000 | 500 | 800 | 900 | 960 | 980 | 990 |
FD Glucose 5 µg/mL (µL) | 0 | 500 | 200 | 100 | 40 | 20 | 10 |
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 extracellular FD-glucose depletion (B) in control (i.e., without insulin) (hatched bars) and insulin-stimulated cells (black bars). FD-glucose uptake was normalized by protein concentrations. Data are shown as % of the value measured in control incubated without FD-glucose (mean SD, n = 8 per condition). Significance was examined by unpaired t-test. (C) Correlation between intracellular and extracellular FD-glucose (%) measures with (squares) or without (circles) insulin in experiments described in (A) and (B). Pearson correlation, P < 0.001.
The authors have nothing to disclose.
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 |