Mitotracker red cmxros probe

Reactive oxygen species (ROS) were detected using 2′,7′-dichlorodihydrofluorescein acetate (H2-DCF; Sigma-Aldrich, St. Louis, MO, USA), and fluorescence intensity was measured according to the fluorescence detection conditions of FITC by using a MACSQuant Analyzer (Miltenyi Biotech, North Rhine-Westphalia, Germany), as already reported [21 (link)].
Moreover, to measure changes in the mitochondrial mass, cells were reacted with 200 nM MitoTracker Red CMXRos probe (Thermo Fisher Scientific, Milan, Italy) for 30 min at 37 °C, according to the manufacturer′s instructions. After being washed twice, cells were treated. Then, 24 h later, they were analyzed by flow cytometry.

Immunofluorescent analysis was performed as already described [21 (link)]. After treatment, cells were adhered to slides by cytospin and subsequently fixed with 4% formaldehyde for 20 min at room temperature. The slides were then incubated overnight at 4 °C with the primary antibody against Nrf2 (anti-rabbit; Santa Cruz Biotechnology, Dallas, TX, USA) and NF-kB (anti-rabbit; Santa Cruz Biotechnology) at a dilution of 1:100. The slides were mounted with medium containing DAPI (4,6-diamidino-2-phenylindole) to visualize nuclei.
To investigate mitophagy, cells were labeled with 200 nM MitoTracker Red CMXRos probe (M7512, Thermo Fisher Scientific, Rodano, Milan, Italy) before the treatment. Cells were subsequently incubated with primary antibody against LC3-II-rabbit (L7543, Sigma-Aldrich, Milan, Italy) at 1:100 dilution. The fluorescent images were obtained using a Zeiss Axio Imager Z1 Microscope with Apotome 2 system (Zeiss, Milan, Italy).

Immunofluorescence for TOM20 and the Mitotracker Green probe (Thermofisher Scientific, Waltham, MA, USA) were used to quantify mitochondrial mass. The Mitotracker Red CMXROS probe (Thermofisher Scientific, Waltham, MA, USA) was used to evaluate morphology. At the end of each treatment, the muscle fibers were washed with Hank’s balanced salt solution (HBSS) and incubated with a Mitotracker Red CMXROS probe (20 nM) or Mitotracker Green (20 nM) in HBSS 1X for 30 min at 37 °C, 5% CO₂. Corresponding fibers were washed with HBSS, fixed with paraformaldehyde 4%, and incubated with the anti-TOM20 antibody (1:100, Cell Signaling, Danvers, MA, USA) overnight. AlexaFluor 488 conjugated anti-rabbit (Thermofisher Scientific, Waltham, MA, USA) was used as the secondary antibody. Images were captured using a confocal microscope Leica SP8 and analyzed using ImageJ software (NIH, Bethesda, MD, USA) with the tool Mitochondrial Network Analysis (MiNA).

Mitochondrial activity was measured in HuH7 and C2C12 cells using a MitoTracker^® Red CMXRos probe (Thermo Fisher Scientific, Waltham, MA, USA). The probe was diluted in DMEM to a final a dye working solution of 100 nM. HuH7 and C2C12 cells were washed twice in PBS before being cultivated in the presence of the probe for 60 min in a cell incubator set to 37 °C and 5% CO₂. Upon incubation, cells were extensively washed in DMEM and once in PBS, and then fixed in 3.7% formaldehyde for 30 min. MitoTracker fluorescence was measured using a Perkin Elmer Envision 2105 Multiplate Reader (Perkin Elmer, Waltham, MA, USA) with the following parameters: λexcitation at 579 nm and λemission at 599 nm. Cells were then permeabilized in 0.1% Triton X-100 in PBS and stained with the nuclear dye DAPI. DAPI fluorescence was measured using a λexcitation at 351 nm and λemission at 450 nm. In order to normalize mitochondrial activity to the total number of cells, Mitotraker fluorescence was normalized to DAPI fluorescence.

Immunocytochemistry was carried out as previously reported [77 (link)]. Briefly, mitochondria were stained with 200 nM MitoTracker Red CMXRos probe (Thermo Fisher Scientific, Milan, Italy) for 30 min at 37 °C, according to the manufacturer’s instructions. Cells were treated with the dye for 30 min at 37 °C, and it was removed after 30 min. At this stage, cells were washed 3 times in phosphate-buffered saline (PBS) to remove the unbound probe. Nuclei were stained by NucBlue (two drops per mL) (Thermo Fisher Scientific, Milan, Italy) for 15 min at 37 °C, according to the manufacturer’s instructions. Finally, cells were treated with lactate 20 mM. For image acquisition, we used Operetta (Perkinelmer, MA, USA), where cells were maintained at 37 °C and images were captured at 24 h after treatment. Data collected were analyzed by Harmony software (Perkinelmer, MA, USA).

To assess oxidative DNA damage, AC16 Cells were incubated with an anti-DNA/RNA Damage (8-oxoG) antibody (Abcam, ab62623), as described previously [31 (link)], and images were acquired using ZEN 2.1 software (Carl Zeiss, Oberkochen, Germany). Co-localization of DNA oxidation and mitochondria was assessed by immunofluorescent analysis of Translocase Of Outer Mitochondrial Membrane (TOM20, proteintech, 11802-1-AP) and 8-oxoG (Abcam, ab62623) in AC16 cells. To explore whether the place where CAV3 interacts with NDUFA10 is located in mitochondria, AC16 cells were labelled with the MitoTracker Red CMXRos probe (Thermo Fisher Scientific, Waltham, MA, USA), an anti-CAV3 antibody (Abcam, ab289544), an anti-NDUFA10 antibody (Santa Cruz, sc-376357), and DAPI (Servicebio, Wuhan, China). The cells were also incubated with an anti-Lamp1 antibody (Abcam, ab208943). Immunofluorescence co-localization images were captured using a laser scanning confocal microscope (Leica, SP8, Germany) and processed using LAS X 3.7.4 software (Leica, SP8, Germany).