Mitotracker red cmxros

Cells were stained with 200 nM MitoTracker® Red CMXRos (MTR) (M9940, Solarbio, Beijing, China) in completed medium for 30 min at 37℃. The samples were then imaged under a confocal laser scanning fluorescence microscope. The excitation/emission wavelengths were 579/599 nm.

For mitochondrial imaging, MitoTracker Red CMXRos (Solarbio, Beijing, China) was used according to the instructions. The adherent cells on coverslips were incubated with 100 nM MitoTracker red for 30 min. Then, the nuclei were stained with fluorescent mounting medium (ZLI-9556, ZSGB-BIO, Beijing, China) on a glass slide. The morphology of mitochondria was observed through TCS-SP8 DIVE (Leica, Wetzlar, Germany) and mitochondria 2D analysis was conducted by Image J Fiji according to the literature [22 (link)].

To detect the expression of mitochondrial cytochrome c, a digitonin-based permeabilization method was employed in this study [52 (link), 53 (link)]. First, 3000 MDA-MB-231, MCF-7 and E0771 cells were seeded on a slide and cultured in a 24-well plate overnight. The cells were treated with 50 ng/mL FGF21 and DOX (1.25 μM for MDA-MB-231 and E0771, 5 μM for MCF-7). The treatment duration was 20 h. After the treatment, the cells were stained with Mitotracker^® red CMXRos (M9940, Solarbio) for 45 min. Following staining, the cells were washed with PBS and fixed with 4% paraformaldehyde for 10 min at 37 °C. To permeabilize the cells, a purified digitonin (0.004%, D5628, Sigma) was used for 2 min at room temperature. The digitonin was prepared according to the manufacturer’s protocol.
Next, the cells were incubated with blocking buffer (3% BSA, 0.2% sodium azide, and 0.1% tween-20 in PBS) for 30 min at room temperature. Subsequently, the cells were incubated with primary antibody (ab110325, Abcam, diluted 1:100) overnight at 4 °C. After the primary antibody incubation, the cells were incubated with Alexa Fluor-conjugated secondary antibodies (ab150105, Abcam) for 1 h at room temperature. Hoechst 33342 was used for counterstaining for 10 min. Finally, the slides were mounted using a mounting medium (S3023, Dako) and analyzed under a confocal microscope (LSM980, ZEISS).

The 3T3-L1 adipocytes were washed with PBS for 3 times, fixed with 4% paraformaldehyde solution for 30 min, and then washed again for 3 times. The fixed samples were permeated with 0.2% Triton X-100 for 20 min and sealed in 5% normal goat serum in PBS for 1 h. The cells were then incubated overnight at 4 °C with the appropriate primary antibodies (1:100 dilution). The next day, the 3T3-L1 adipocytes were washed and incubated at room temperature with goat anti-rabbit secondary antibody or goat anti-mouse secondary antibody (1:1000 dilution) for 2 h and washed with PBS for 3 times. The nuclei were stained with DAPI. For mitochondrial detection, the 3T3-L1 adipocytes were incubated with 100 nM MitoTracker Red CMXRos (Solarbio, Beijing, China) in the dark at 37 °C for 30 min. Fluorescence images were taken using a laser confocal microscope (SP8, Leica, Germany). At least four images per section were acquired for quantification, and the mean intensity were evaluated using ImageJ.

Mitophagy was visually assessed using the colocalization of mitochondria and lysosomes [25 (link)]. To observe mitochondria, HCCLM3 cells were dyed with 200 nmol/L MitoTracker Red CMXRos (M9940, Solarbio) for 30 min at 37 °C. The cells were then stained for 60 min with 100 nmol/L LysoTracker Green DND-26 (L7400, Solarbio) to detect lysosomes. After experimenting with new cultural mediums. Images were captured using an Olympus FV3000 confocal microscope (Olympus). Fluorescences were stimulated at 578 nm (Red) and 488 nm (Green) (Green). Images were analyzed using Fluoview FV31S-SW software (Olympus) to determine MitoTracker and LysoTracker colocalization.

HADSCs were inoculated in six-well plates, and when the proliferation of hADSCs was observed to be confluent to about 40%, the medium was changed to the corresponding induction medium. At the end of induction differentiation, MitoTracker® Red CMXRos (200 nM, 1 ml; Solarbio, China) staining solution was added and incubated in an incubator at 37 °C 20 min. After the incubation, they were quickly observed under a fluorescent microscope and photographed.

MitoTracker Red CMXRos (200 nM, Solarbio) were utilized to stain the mitochondria of EA.hy926 cells for 30 min after P. gingivalis treatment. After washing, fixing, permeabilizing and blocking, the cells were treated with the rabbit anti‐p‐Drp1 (Ser616) antibody (1:400, CST, MA, US) at 4°C overnight. Then, p‐Drp1 (Ser616) was detected using a secondary goat anti‐rabbit antibody (1:100, Solarbio). The CLSM was used to capture the images.