Mitotracker red probe

The production of reactive oxygen species (ROS) was assayed by quantifying the oxidation of 5-6-chloromethyl-2,7-dichlorodihydro-fluorescein diacetate (CM-H₂DCFDA, Invitrogen Corporation) on a plate fluorescence reader at 520–595 nm. Results were normalized against DAPI fluorescence, as described previously [19 (link)]. ROS production was also assessed by the oxidation of nitro blue tetrazolium (NBT, Sigma-Aldrich), as quantified by the absorbance at 560 nm and normalized against the protein content. To evaluate mitochondrial dysfunction, we used the cationic dye tetrachloro-tetra-ethyl-benzimidazolyl-carbocyanine iodide (JC-1) to evaluate mitochondrial membrane potential, and the MitoTracker Red probe (both from Invitrogen Corporation) to measure mitochondrial mass, as described previously [19 (link)]. Briefly, cells were cultured in 96-well plates and incubated with JC-1 (4 µg/mL) or MitoTracker (50 nmol/L) for 2 h at 37 °C in the dark. The results were quantified on a plate fluorescence reader at 520–595 and 485–535 nm for JC-1 aggregates and monomers, respectively, and at 575–620 nm for MitoTracker.

Fibroblast cells were cultured in glass‐bottomed dishes. The mitochondria was visualized using MitoTracker red probe (Invitrogen). Then cells were washed with ice‐cold phosphate‐buffered saline (PBS) three times and were fixed with 4% paraformaldehyde for 15 min at room temperature. Fluorescence images were captured by ZEISS LSM900 confocal system.

The cationic dye tetra-chloro-tetra-ethyl-benzimidazolyl-carbocyanine iodide (JC1) was used to evaluate the mitochondrial membrane potential, and the Mitotracker Red probe (both from Invitrogen Corporation) was used to measure the mitochondrial mass. The production of ROS was assessed by the oxidation of 5–6-chloromethyl-2,7-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA) (Invitrogen Corporation). Cells cultured in 96-well plates were incubated with JC1, Mitotracker, or CM-H₂DCFDA for 2 hr at 37°C. The fluorescence was quantified on a plate reader at 520–595 nm for JC1 aggregates, 485–535 nm for JC1 monomers, 575–620 nm for Mitotracker, and 485–520 nm for CM-H₂DCFDA. The results were normalized against DAPI fluorescence.

The cationic dye JC-1 (tetrachloro-tetra-ethyl- benzimidazolyl-carbocyanine iodide) evaluated the mitochondrial membrane potential, and the MitoTracker Red probe (both from Invitrogen Corporation,) measured mitochondrial mass. Cells, cultured in 96-well plates, were incubated with JC-1 (4 μg/mL), or Mitotracker (50 nmol/L) in DMEM, for 60 min at 37 °C in the dark. Quantification was performed on a plate fluorescence reader (Spectrafluor Plus, Tecan-France, Trappes, France) at 595 and 530 nm (JC-1 aggregates and monomers, respectively) or 630 nm (MitoTracker). The production of ROS was assessed by the oxidation of 5-6-chloromethyl-2,7-dichlorodihydro-fluorescein diacetate (CM- H₂DCFDA) (Invitrogen Corporation) or the reduction of nitroblue tetrazolium (NBT) (Sigma-Aldrich) (Hernandez-Vallejo et al., 2013 (link)). Results were normalized to cell protein content. Superoxide dismutase (SOD) activity was evaluated using a commercially available kit (Sigma-Aldrich).

The HeLa cell line was purchased from the ATCC and cultured in DMEM (GIBCO) supplemented with 10% FCS (Gibco). The DF1 cell line (UMNSAH/DF-1, ATCC CRL-12203, kind gift of Gaël Orieux, UPMC, Institut de la Vision, Paris, France) was cultured in DMEM supplemented with 5% FCS and 5% chick serum (Gibco).
Mitochondria were labeled by incubation of the cells for 40 min with the Mitotracker Red probe (Invitrogen) at 0.1 μM in culture medium. Cell nuclei were detected by Hoechst (Sigma) post-fixation labeling.

Cells (1 × 10⁵) were plated on glass bottom microwell dishes (NEST, Jiangsu, China; #801002). The next day, culture medium was removed, cells were washed and stained with 100 nM MitoTracker red probe (Molecular Probes, Eugene, OR, USA) for 30 min. Fluorescence was observed by using an LSM880 confocal microscope (Zeiss, Oberkochen, Germany) equipped with a 63 × oil objective.