Mitochondrial mass determination

MitoTracker Green FM (#9074; Cell Signaling Technology, Danvers, MA, USA) was used to determine the mitochondrial mass via flow cytometry analysis. Cells were incubated with 200 nM MitoTracker in the dark at 37°C for a duration of 15 min. Mitochondrial mass per cell was subsequently determined by quantification of mean fluorescence intensity (MFI) green using a FACS Canto II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

For microscopic imaging, CD4⁺ and CD8⁺ T cells were incubated as described above. After two additional washing steps, lymphocytes were seeded in µ‐slides 2 × 9 well (Ibidi, Gräfelfing, Germany). Images of 10 randomly chosen microscopic fields per well at 200× magnification and 3× digital zoom were acquired on a LEICA‐TCS SP5 Confocal Microscope (Leica, Wetzlar, Germany) using Leica application suite AF software, version 2.7.