Bodipy fl c16

To check the BODIPY FL C₁₆ uptake in control or ↑[H⁺]-treated T cells, T cells were cultured with freshly dissolved 1 μM BODIPY FL C₁₆ (Invitrogen) for 1 h, then washed twice with PBS prior to surface staining. To further explore the metabolic dependence in different treatment groups, experiments were performed using the SCENITH (single cell energetic metabolism by profiling translation inhibition) method^{42 (link)}. Briefly, T cells were seeded in 96-well plates at 1 × 10⁶ cells/ml. Then cells were treated with control (Ctrl), 2-deoxy-d-glucose (final concentration 100 mM), oligomycin (final concentration 5 μM), or a mix of the inhibitors at the final concentrations for 40 min at 37 °C. Puromycin (final concentration 10 μg/ml) was added during the last 30 min. After puromycin treatment, cells were washed with cold PBS and pre-stained with Live/Dead Fixable Dead Cell Stain Kit and antibodies against surface markers. The staining of intracellular puromycin was followed by the staining process for intracellular transcription factors.

Commercially available compounds were used without further purification unless otherwise stated. BODIPY-FL-C₁₆ was purchased from Thermo Fischer Scientific (99%) (Netherlands). 1,3-diolein was purchased from Sigma Aldrich (≥ 99%). DMEM/F-12 was purchased from ThermoFischer (Waltham, MA).
All HPLC purifications (1.0 mL/min, solvent A; 0.1% TFA in water, solvent B; CH₃CN, 50°C) were performed on a Shimadzu UFLC HPLC system equipped with a DGU-20A₅ degasser, a SPD-M20A UV detector, a LC-20AT pump system, a CBM-20A communication BUS module, a CTO-20AC column oven, and a Scan-RAM radio-TLC/HPLC-detector from LabLogic using an Aeris™Widepore column (XB-C18, 3.6 μm, 4.6 mm × 250 mm) for the BDP-FA or an Aeris™Widepore column (C4, 3.6 μm, 4.6 mm × 250 mm) for the Bodipy-triglyceride (BDP-TG). ESI-MS was performed on a Applied Biosystems SCIEX API 150 EX electrospray ionization quadrupole (ESI-Q) mass spectrometer with the method of McAnoy et al. [34 (link)]. Briefly, 0.1M aqueous ammonium acetate solution was added to the probe to observe the ammonium salt in the MS.
¹H-NMR spectra were carried out on a Bruker UltrashieldTH 400 plus at 400 MHz. Tol-d₈ was used as solvent with TMS as internal standard. Chemical shifts are reported in parts per million (ppm) relative to the internal standard.

2-NBDG and BODIPY FL C₁₆ uptake assays were performed as previously described (21 (link)). Briefly, PBMCs were incubated in glucose-free RPMI (Thermo Fisher Scientific) supplemented with 50 μM 2-NBDG (Biomol, Hamburg, Germany), PBS supplemented with 12.5 μM BODIPY FL C₁₆ (Thermo Fisher Scientific) or RPMI containing 10% (v/v) FBS supplemented with 100 nM MitoTracker Green and 12.5 nM MitoTracker Deep Red (Thermo Fisher Scientific) for 30 min at 37°C, 5% (v/v) CO₂. Subsequently, surface staining was carried out as above. Cells were washed with PBS, fixed with 0.5% (w/w) PFA and then acquired on a BD LSRFortessa flow cytometer (BD Biosciences).

Cells were first seeded onto 12-wells at a density of around 200k/well and medium was changed every 2-3 days. On day 30 ± 3, CMs were stained with the MitoTracker deep red (MTDR) dye (200 nM; Thermo Fisher Scientific) and a fluorescently labelled palmitate analogue BODIPY FL C16 (10 µM; Thermo Fisher Scientific) for 25 min. The cells were dissociated, washed and resuspended in Hank’s buffered saline solution (HBSS) containing 10 µM Y27632 inhibitor, 25 mM HEPES (Thermo Fisher Scientific), and 5% FBS. DAPI staining (2.5 µM) was also included to help gate out dead cells during flow cytometry. Cells were then loaded onto the Attune NxT Flow Cytometer (Thermo Fisher Scientific) and analyzed. BODIPY FL C16 and MTDR staining were calculated as the median FITC-A and APC-A, respectively.