Mitotracker green fm

Mitochondrial mass, membrane potential and FA uptake of freshly sorted or cytokine-activated ILC2s were assessed by staining cells with 50 nM MitoTracker Green FM (Thermo Fisher), 25 nM TMRM (Sigma-Aldrich) and BODIPY FL-C₁₆ (Thermo Fisher), respectively, for 30 min at 37 °C and 5% CO₂. Cells were washed twice in cold 1× PBS, stained with surface antibodies and analyzed by FACS. For confocal microscopy, cells were stained at 37 °C for 30 min with 300 ng ml^–1 of Hoechst H33342 (Life Technologies) to stain nuclei, 100 nM MitoTracker Green FM to stain mitochondria and 25 nM TMRM to asses mitochondrial membrane potential (non-quenching mode, TMRM maintained in the cell medium). Cells were plated in a 384-well plate (40,000 cells per well), and image acquisitions of multiple fields per well were performed on an automated confocal microscope (OPERA QEHS, Perkin Elmer) using ×60 objectives, excitation lasers at 405, 488 and 561 nm and emission filters at 450, 540 and 600 nm, respectively. Confocal images were transferred to the Columbus Image Data Storage and Analysis System (Perkin Elmer) for high content analyses as previously reported^{57 (link)} and used the standard deviation/mean approach^{58 (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.

To detect superoxide levels in the mitochondria, MitoSOX Red (Molecular Probe, Thermo Fisher Scientific, M36008), MitoTracker Green FM (Molecular Probe, Thermo Fisher Scientific, M7514), and Mitochondrial superoxide detection kit (Abacm, Cambridge, UK) were used according to the manufacturer’s protocol, respectively. Briefly, the cells were incubated with UPA (10 μmol/L) or GBH (10–200 μg/ml) for 48 h and then loaded with MitoSOX Red (5 μmol/L) and MitoTracker Green FM (100 mmol/L) in Hanks’ Balanced Salt Solution (HBSS) (GIBCO, Thermo Fisher Scientific, 14175095) at 36°C for 10 min. After the cells were rinsed with PBS, each slide was covered with ProLong^® Gold anti-fade mountant with DAPI (Life Technologies, Thermo Fisher Scientific, P36935).
For mitochondrial superoxide detection, hUtMCs were seeded 3 × 10⁴/ml in a 6-well plate and were treated with UPA (10 μmol/L), GBH (10 – 200 μg/ml), and Mito-TEMPO (50 μmol/L) for 48 h. After treatment, loaded with loaded with MitoROS 580 at 37°C for 1 h. A fluorescent intensity was measured at an excitation wavelength of 540 nm and an emission wavelength of 590 nm using a SpectraMax Gemini XPS/EM fluorescence plate reader (Molecular Devices, LLC., San Jose, CA, USA). And, the fluorescence signal was measured using fluorescence microscope (Olympus, Tokyo, Japan).

For JC-1 staining assay (Cat. T3168, ThermoFisher Scientific), the EOC of 2 × 10⁵ cells per tube were stained by 2 μM JC-1 for 30 min at 37 °C CO₂ incubator. The cells were analyzed with a FACScan flow cytometer (BD Biosciences, San Jose, CA) for quantifying 488 nm excited fluorescence signals at 574 nm (JC-1 aggregate, red) and 520 nm (JC-1 monomer, green). EOC cells were seeded in 6-cm culture dishes at a density of 1 × 10⁵ cells. A mitochondrial membrane potential assay was performed using the Membrane Potential Assay Kit (II) (#13296S, Cell Signaling Technology, Danvers, MA, USA) and MitoTracker Green FM (Thermo Fisher Scientific, Rockford, IL, USA) according to the manufacturer’s instructions. Briefly, cells were stained with 25 nM TMRE and 100 nM MitoTracker Green FM (ThermoFisher Scientific, Rockford, IL, USA) for 15 min in a 5% CO₂ incubator. Then, the cells were analyzed with a FACScan flow cytometer (BD Biosciences, San Jose, CA). The excitation wavelength is 488 nm, and the emission wavelengths are 578 nm (TMRE, red) and 516 nm (Mitotracker, green). Fluorescence-activated cell sorting (FACS) data were analyzed for dot plots and histogram plots using Flowing Software version 2.5.1 (Turku bioimaging, Turku, Finland). Fluorescent images of the cells were acquired using an EVOS FL cell imaging system (Thermo Fisher Scientific, Rockford, IL, USA).

MSCs were stained with MitoTracker Green FM (Invitrogen, Carlsbad CA, USA) to quantify functional mitochondrial mass. Briefly, 48 h prior to staining 1 × 10⁴ cells were seeded using 35 mm glass bottom culture dishes, containing 14 mm microwell (MatTek, MA, USA). Cells were incubated for 30 min with DMEM supplemented with 80 nM MitoTracker Green FM, followed by 5 μM CellTracker Red CMTPX solutions to stain the cytoplasm (Invitrogen), and Hoechst (Invitrogen) to define cell nucleus. Cells were observed in vivo, and 2D and 3D images were collected by confocal microscopy using an Olympus FV1000 with an excitation/emission range of 400/545 for MitoTracker Green FM and 577/602 nm for CellTracker Red CMTPX. Z-stack parameters were as follows: ~15 z-axis slices at ~0.50 μm intervals with a final Z-stack thickness of ~7.5 μm. Mitochondrial mass was determined by data obtained from confocal microscopy in voxel units.

To characterize the endocytosis process by which MSCs internalize cardiac mitochondria, human MSCs were exposed to cardiac mitochondria previously labeled with MitoTracker Green FM (40 nM, Invitrogen, Waltham, MA, USA, Cat#M7514) in the presence of the dynamin-dependent, clathrin-mediated endocytosis inhibitor dynasore (50 mM, Santa Cruz Biotechnology, Dallas, TX, USA, Cat#sc-202592). After 24 h of treatment, the MitoTracker Green FM (40 nM, Invitrogen, Waltham, MA, USA, Cat#M7514) fluorescence of MSCs was analyzed with flow cytometry or LSM800 confocal microscopy (Zeiss, Oberkochen, Germany).

Splenic cells were resuspended with pre-warmed (37 °C) RPMI-1640 medium in the presence of 20 nM MitoTracker^® Green FM (Invitrogen) or TMRE (Invitrogen), and then cultured at 37 °C for 30 min in the dark. Surface marker were then stained for detection via flow cytometry. For MitoSOX staining, instead of MitoTracker^® Green FM or TMRE, 5 μM MitoSOX^™ (Invitrogen) was used. Following culture at 37 °C for 10 min in the dark, cells were then stained for surface markers for assessment by flow cytometry.