Mitotracker green fm

For mitochondrial staining, 100,000 cells were seeded in 12-well plates. The day after, MitoTracker Green FM (Cell Signaling Technologies, #9074S) was diluted to 400 nM directly into fresh media and added to the cells, which were incubated for 30 min at 37 °C. After incubation, media was changed, and live cells were imaged using IncuCyte-Zoom.

Hydrogen peroxide, 2′,7′-dichlorfluorescein-diacetat (DCFH-DA), Mito-TEMPO, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lipoic acid, and transferrin were obtained from Sigma-Aldrich (Darmstadt, Germany). TUDCA was bought from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). MitoTracker™ Red CMXRos; MitoSOX™ Red; DAPI; Goat anti-Mouse IgG (H + L), Superclonal™ Recombinant Secondary Antibody, Alexa Fluor™ 488; and goat anti-Rabbit IgG (H + L), Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488 were purchased from Thermo Fisher Scientific (Karlsruhe, Germany). MitoTracker^® Green FM was obtained from Cell Signaling Technology (Leiden, The Netherlands). Mouse anti-Ki-67 antibody was bought from BD Sciences (BD Pharmingen™, Heidelberg, Germany).
Human embryonic kidney (HEK) 293 cells were kindly provided by Dr. Rolf Sprengel (Department of Molecular Neurobiology, Max Planck Institute for Medical Research, Heidelberg, Germany). Human hepatocellular carcinoma cell line HepG2 was a kind gift from Prof. Stephan Urban (Department of Infectious Diseases, Molecular Virology, Centre for Integrative Infectious Disease Research (CIID), Heidelberg University, Heidelberg, Germany).

Erythroblasts and iPSCs were washed and resuspended in FC buffer (HBSS w/o calcium and magnesium + 0.5% BSA) and incubated with PE Mouse Anti-Human CD44 antibody (1:25, BD, 550989), FITC Mouse Anti-Human CD71 antibody (1:50, BD, 555536), or 7-AAD (Thermo Fisher, A1310) for 30 min at 4 °C. Mitochondria were stained with Mitotracker Green FM (100 nm, Cell Signaling, 9074) and active mitochondria with TMRM (100 nm, Thermo Fisher, T668) for 30 min at 37 °C. Cells were detected by flow cytometry using a LSRFortessa Cell Analyzer (BD, USA). Flowjo software (BD, USA) was used for data analysis.

U2OS cells were cultured on glass coverslips and incubated with 20 μM Mito-switch and 0.1% Pluronic F-127 (Beyotime, ST501) for 4 h at 37 °C. For colocalization experiments, 100 nM MitoTracker Green FM (Cell Signaling Technology, 9074 S) was added for 30 min at 37 °C. For photoswitching experiments, U2OS cells were incubated with 40 μM Mito-switch and 0.1% Pluronic F-127 for 4 h at 37 °C. For photostability experiments, U2OS cells were incubated with 40 μM Mito-switch and 0.1% Pluronic F-127 for 6 h at 37 °C.

MitoTracker™ Green FM purchased from Cell Signaling Technology was dissolved in DMSO to prepare a 1 mM stock solution. SH-SY5Y cells were treated as described in Section 2.1. MitoTracker™ Green FM was mixed with the fresh medium at a concentration of 500 nM. After 20 min of reaction at 37 °C, the samples were analyzed by flow cytometry analysis (Excitation: 490 nm, Emission: 516 nm) or fluorescence microscopy to observe the content of cellular mitochondria.

Mitochondrial mass and membrane potential were assessed using MitoTracker® Green FM (0.1 μM; Cell Signaling Technology, Danvers, MA) and tetramethylrhodamine ethyl ester (TMRE; 0.05 μM, ThermoFisher), respectively, according to the manufacturers' instructions. To assess viability, cells were stained with 7-amino-actinomycin (7-AAD; (BioLegend, San Diego, CA) in accordance with the manufacturer's instructions. Data were acquired with a Fortessa flow cytometer (BD Biosciences, San Jose, CA) and analyzed using FlowJo (TreeStar, Ashland, OR).