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

Protocolo

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 ready-to-use cell culture reagents: Trypsin-EDTA, phosphate-buffered saline (PBS), and glucose-free and phenol red-free Dulbecco's Modified Eagle Medium (hereafter, glucose-free DMEM).
3. Use 20 mL of radioimmunoprecipitation assay (RIPA) lysis buffer.
   NOTE: In the following sections, extracellular depletion and intracellular uptake of different FD-glucose doses are compared in 3T3-L1 fibroblasts with and without insulin stimulation (Figure 1).

2. 3T3-L1 cell culture and maintenance

1. Culture 3T3-L1 mouse fibroblasts by plating 1 x 10⁶ cells diluted in 20 mL of Medium 1 in two 96-well plates. Plate 100 μL of cell suspension per well, ensuring to maintain homogeneity of this suspension by mixing. Grow cells for 48 h in a 37 °C incubator (5% CO₂ and 95% humidity) without changing media until about 70%-80% confluency.
2. Maintain cells confluent and fasted by culturing them in the same medium for 48 h. Vary the incubation time from 24-72 h depending on the desired fasting catabolic state.

3. Starvation of 3T3-L1 cells

1. Decant media from cells in the 96-well plates. Absorb the remaining fluid using sterile paper towels.
2. Rinse the 96-well plate with 100 μL of PBS per well and absorb the remaining fluid with sterile paper towels.
3. Add 100 μL of glucose-free DMEM per well and incubate for 40 min. Adjust the time of incubation depending on cell type, stimuli, and desired level of fasting.
   NOTE: The time of incubation may require optimization depending on the season.

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.

1. Use eight replicates (one row in a 96-well plate) for each stimulation condition. Therefore, prepare 1 mL for each stimulation condition (specified in Table 2 and below).
2. Use eight stimulation conditions without insulin: FD-glucose of 2.5 μg/mL, 1 μg/mL, 0.5 μg/mL, 0.2 μg/mL, 0.1 μg/mL, 0.05 μg/mL, and 0 μg /mL (control without FD-glucose) in one 96-well plate.
3. Use eight stimulation conditions with insulin (10 μg/mL, final concentration): FD-glucose of 2.5 μg/mL, 1 μg/mL, 0.5 μg/mL, 0.2 μg/mL, 0.1 μg/mL, 0.05 μg/mL, and 0 μg/mL (control without FD-glucose) in another 96-well plate.
4. To prepare stimulation conditions, dilute 1 μL of 5 mg/mL FD-glucose working solution (Table 1) in 999 μL of glucose-free DMEM to obtain 1 mL of working stock solution (1:1000 dilution or 5 μg/mL).
   1. Using this working stock solution, prepare 1:2000, 1:5000, 1:10,000, 1:25,000, 1:50,000, and 1:100,000 dilutions of FD-glucose to obtain 2.5 μg/mL, 1 μg/mL, 0.5 μg/mL, 0.2 μg/mL, 0.1 μg/mL, and 0.05 μg/mL (Table 2) in 2 mL tubes immediately before experiments in a biosafety cabinet without lights.
5. Repeat step 4.4 and add 1 μL of insulin (10 mg/mL, final concentration) to each of the conditions specified above.
   NOTE: The final concentration of insulin in these samples is 10 μg/mL = 1.7 nmol/mL. To achieve homogenization, add insulin to the tubes before adding other solutions.

5. Treating starved 3T3-L1 cells

1. After 40 min of incubation, decant the glucose-free DMEM from each well in 96-well plates and absorb the remaining fluid with sterile paper towels.
2. Add 100 μL (per well) each from the various treatment conditions described above to wells along one column, and 100 μL (per well) from the same treatment condition to the wells along the row (eight replicates) in both 96-well plates. Label the plates that utilized the conditions with and without insulin. Add glucose-free DMEM alone to the control wells (n = 8).
3. Incubate the plate for 40 min in a cell culture incubator (37 °C, 5% CO₂, and 95% humidity) in the dark.

6. Extracellular and intercellular measurements for stimulated 3T3-L1 cells

1. After the cells have been stimulated for 40 min, transfer the stimulation media from both the plates into new 96-well plates maintaining the same experimental layout.
2. Decant any remaining solution from the original stimulated 96-well plates with cells on sterile paper towels.
3. Optionally, wash cells with PBS. Remove PBS and decant any remaining solution on sterile paper towels.
4. Add 100 μL of the RIPA lysis buffer to each well. Optionally, add protease inhibitor to the RIPA buffer to protect proteins. Place plates containing RIPA in a shaker for 30 min.
5. Using a microplate reader (Table of Materials), measure fluorescence at excitation (Ex) and emission (Em) wavelengths of 485 and 535 nm, respectively, first in the medium containing extracellular FD-glucose, and then, the plate with RIPA-lysed cells at the end of 30 min incubation (see step 6.4).

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	1:2x103	1:5x103	1:1x104	1:2.5x104	1:5x104	1:1x105
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

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Resultados

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.

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Materiales

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