Mitotracker deep red

MitoTimer in adult muscle fibers was performed as previously published (3 (link), 9 (link)–11 (link, link)). For C2C12 cells transfected with pmitoAIP, cells on glass plates were stained with 400 nM MitoTracker Deep Red in DMSO (Sigma) for 30 min and transferred to live cell imaging chambers (Invitrogen) in phenol-red–free DMEM with 20% FBS. Images were acquired at 100× magnification (Olympus Fluoview FV1000) using red (via 543-nm laser) and far red (via 635-nm laser) channels through a tetramethylrhodamine (TRITC) (Ex/Em 555/580 nm) and Alexa Fluor 647 filters (Ex/Em 649/666 nm), respectively. Mitochondrial content as a percentage of cell volume was analyzed via mitochondria morphology macro in Image J (80 (link)).

To explore the interaction of mitochondria and CD4⁺ T cells, the platelets derived mitochondria were stained with MitoTracker Deep Red FM (100 nM) (Thermo Fisher Scientific, Waltham, MA, USA) and cocultured with MitoTracker Green FM (100 nM) (Thermo Fisher Scientific, Waltham, MA, USA) labeled CD4⁺ T cells for 2 h, at room temperature. Hoechst 33,342 (Sigma, Saint Louis, MO, USA) was used to stain the nuclei. Briefly, the purified CD4⁺ T cells were initially labeled with MitoTracker Green FM (100 nM) (Thermo Fisher Scientific, Waltham, MA, USA) and Hoechst 33,342 (Sigma, Saint Louis, MO, USA), and then planted in the 96-well tissue culture-treated plates at 2 × 10⁵ cells/well with serum-free culture medium X-VIVO 15 in the presence or absence of MitoTracker Deep Red-labeled platelet-derived mitochondria. The interaction between mitochondria and CD4⁺ T cells was directly observed and photographed under a confocal microscope with Nikon A1R confocal microscope on Nikon Eclipse Ti2 inverted base at room temperature.