Mitosox red mitochondrial superoxide indicator

Pulmonary arterial smooth muscle cells were incubated with 5.0 μM MitoSOX Red Mitochondrial Superoxide Indicator (Yeasen, China) for 15 min to detect mitochondrial superoxide production using a fluorescent microscope (Olympus, Japan).

To detect cytosolic ROS, MLE-12 cells were incubated with 10 μM 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) (Nanjing Jiancheng, Nanjing, China) for 1 h and then washed with PBS. The fluorescence intensity was measured by a fluorescence spectrophotometer (the wavelength was 485 nm and the emission wavelength was 530 nm). For the detection of mitochondrial ROS, MLE-12 cells were stained with 5 μM of MitoSOX Red Mitochondrial Superoxide Indicator (Yeasen, Shanghai, China) for 10 min. After staining, cells were washed with PBS, and then cells were counterstained with DAPI for 10 min. The stained cells were observed and photographed on a confocal microscope.

HK2 cells were incubated with MitoSOX Red Mitochondrial Superoxide Indicator (YEASEN, Shanghai, China), then visualized and imaged under a confocal microscope (LSM880, ZEISS, Germany).

A membrane-permeable fluorescent probe, 2′, 7′-dichlorofluorescin diacetate (DCFH-DA), and a fluorescence microscope (ECLIPSE NI, NIKON, Japan) were used to detect intracellular ROS. The fluorescence is detected at 525 nm after excitation at 488 nm. The concentration of mitochondrial ROS was detected utilizing MitoSOX Red Mitochondrial Superoxide Indicator (40778ES50, Yeasen, China) and fluorescence microscope (ECLIPSE NI, NIKON, Japan). The fluorescence is detected at 580 nm after excitation at 510 nm.

Cells were cultured and treated with tyrosol as described above. Mitochondrial membrane potential and ROS were stained using MitoTracker Red CMXRos and MitoSOX Red Mitochondrial Superoxide Indicator (both from Yeasen Biotech, China), respectively. Cells were labeled with MitoTracker Red CMXRos (final concentration, 100 nM) or MitoSOX Red (final concentration, 3 μM) at 37°C for 30 min. The cells were then fixed with 4% paraformaldehyde for 15 min, followed by nuclei staining using DAPI (Beyotime). Images were taken with DMI6000B (Leica) and fluorescence intensity was measured using ImageJ software. The results were expressed as the mean of relative fluorescence intensity per cell.

Predominant ROS in mitochondria was determined using MitoSOX Red mitochondrial superoxide indicator (Yeasen, Shanghai, China). Briefly, HK-2 cells were treated with strategies as previously described; cells were incubated with 5 μM MitoSOX reagent working solution at 37°C for 15 min. Cells were then treated with DAPI solution for 15 min and imaged randomly at ×200 magnification using a fluorescence microscope. Mitochondrial ROS generation was shown as the fluorescence intensity of the red color. MitoSOX-positive cell ratio was calculated in 6 randomly selected fields per sample, and each experiment was repeated in triplicate.
Intracellular accumulation of ROS was evaluated using an oxidation-sensitive DCFH-DA kit (Sigma, the United States). Briefly, HK-2 cells were treated with strategies as previously described and then incubated with DCFH-DA (10 μmol/L) in dark for 1 h. The cells were washed with DMEM/F12 culture medium and collected in PBS. The fluorescence of cells was measured by flow cytometer (Beckman, the United States), using 488 nm excitation and 525 nm emission wavelengths. Each experiment was repeated in triplicate.

The level of intracellular ROS was detected by using an (ROS) assay kit. After being washed by PBS, cells were re-suspended in PBS with 10 µM of DCFH and incubated for 40 min at 37 °C. Then, the cells were collected and washed twice with PBS. The level of ROS was analyzed by flow cytometry at a wavelength pair of 488/525 nm.
Mitochondrial ROS was measured by using mitoSOX red mitochondrial superoxide indicator (Yeasen Biotech, Shanghai, China). This fluorogenic dye can selectively enter the mitochondria of living cells, in which it is oxidized by superoxide anions. After treatment, cells were harvested and washed twice with PBS. The cells were stained with 2 μM of mitoSOX at 37 °C for 10 min, followed by wash with PBS, and then analyzed by flow cytometry wavelengths 510/580 nm.
The intracellular Ca²⁺ was measured by using Fluo-3 AM (Beyotime Institute of Biotechnology, Shanghai, China). After treatment, cells were harvested and washed twice with PBS. The cells were stained with Fluo-3 AM at 37 °C for 30 min without light, and then used immediately for detection by flow cytometry.