2 nbdg

T98G cells (1.0 × 10⁵ cells/well) were plated in a 24-wells plate (Corning flat bottom tissue culture treated plates, Corning Life Sciences, Tewksbury, MA, USA). After 24 h, the cell culture medium was removed, and cells were treated with 100 µg/mL of 2-NBDG (2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-deoxyglucose, Thermo-Fisher Scientific) alone or in combination with GH, or phloretin, in 400 µL of DMEM glucose free, supplemented with sodium pyruvate, glutamax, p/s, and 0.5% FBS. Cells were incubated at 37 °C with 5% CO₂ for 1 h. Subsequently, cells were trypsinized and washed with 1× FACS buffer. Cells were acquired with a BD FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA, USA). Forward Scatter (FSC) vs. Side Scatter (SSC) plots were used to exclude dead cells and nonspecific cellular debris. The mean fluorescence intensity (MFI) of 2-NBDG was measured, and the data were analyzed using the CellQuest Pro software v. 5.1 (BD Biosciences, Franklin Lakes, NJ, USA).

CD4⁺ cells were cultured in RPMI medium in anti-CD3 coated plates (0.5 mg/mL) and activated with IFNγ (50 ng/mL) and YM155 (0 and 10 nM) for 24 h. Cells were washed and starved in glucose-free RPMI-medium for 2 h and then supplemented with 2-NBDG (100 μM, Abcam). 2NBDG uptake was registered after 30 min using flow cytometry (Verse, BD) and quantified as the ratio of mean fluorescence intensity to baseline.

First, the cells were incubated and grown in DMEM (Life Technologies, United States) without the additional FBS and glucose for 24 h to a monolayer confluence. Later, the medium was removed and cells were washed 3× with PBS Krebs ringer phosphate buffer with 50 mM glucose and 200 μM 2-NBDG (2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose) (Thermo Fisher Scientific, United States) was added to the cell layer. Cells were incubated with and without 50 mM insulin for 30 min. The cells were harvested and the 2-NBDG uptake was measured by flow cytometry using a FACSCalibur^TM Cytometry (BD Biosciences, United States). Approximately, 10,000 events were collected for each analysis. Data from the experiments were analyzed using the CellQuest software (BD Biosciences, United States).

Cells were plated onto 6-well plates at a density of 6 × 10⁵ cells/well and allowed to grow until 80% confluence. Then, the cells were exposed to 10 µM 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2NBDG, Thermo Fisher Scientific) in DMEM without glucose under treatment conditions for 4 h. Cells were treated with 5 µM apigenin as a positive control. After treatment, both adherent and suspended cells were collected, washed in DPBS, and exposed to 5 µM 7-aminoactinomycin D (7AAD; BD Biosciences) for 15 min at room temperature protected from light. The determination of viable and positive 2NBDG cell percentages was evaluated by flow cytometry (Accuri C6 Plus Flow Cytometer—BD Biosciences), collecting 50,000 events for each condition.