Mitotracker red cmxros

Live HAECs exposed to either static (STT) or LSS were incubated with 200 nM pre-warmed MitoTracker Green FM or MitoTracker Red CMXRos (Molecular Probes) solution at 37°C for 30 min. After removal of the incubation solution, cells were washed three times with pre-warmed PBS and then mounted in Hank's balanced salt solution. For quantitative analyses, more than 100 images per each group were acquired using an epi-fluorescence upright microscope with a 63x objective oil lens. For MitoTracker Green FM staining, excitation/emission wavelengths were set at 470/525 nm (FL filter Set 38, Zeiss), and for MitoTracker Red CMXRos staining, excitation/emission wavelengths were set at 587/647 nm (FL filter Set 64HE). Images were initially acquired using an AxioCam MRm and AxioVision image processing system (Zeiss), and the fluorescence intensities were assessed using Image J software (NIH).

MitoTracker Red CMXRos (C1049B, Beyotime) was used to detect mitochondrial activity. According to the manufacturer’s instructions, the cells were incubated with a culture medium containing 20 nM MitoTracker Red CMXRos for 30 min at 37 ℃ in the dark and then observed and captured with a fluorescence microscope (ZEISS Vert. A1) after changing the fresh culture medium.

Cell were washed with PBS and fixed with 4% paraformaldehyde supplemented with 0.25% Tris-X100 at room temperature for 10 min. After blocking with PBS supplemented with 5% BSA for 2 h at room temperature, cells were incubated with Mito-Tracker Red CMXRos (C1035, Beyotime), NLRP3 (sc-66846, 1:500, Santa Cruz, USA), at 4° C overnight. Cells were incubated with secondary peroxidase conjugated goat anti-rabbit IgG (1:100, Santa Cruz Biotechnology) antibody for 2 h at room temperature, after washing with PBST for 15 min. Cells were stained with DAPI for 15 min at darkness, after washing with PBST for 15 min. Cell samples were observed using fluorescence microscope (Zeiss Axio Observer A1, Germany).
Next, cells were washed with PBS and stained with Mito-Tracker Red CMXRos for 30 min. Cells washed with PBST for 15 min and stained with DCFH-DA for 30 min. Cell samples were observed using fluorescence microscope (Zeiss Axio Observer A1, Germany) after washing with PBST for 15 min.
Cell were washed with PBS and fixed with 4% paraformaldehyde for 15 min. Cells were stained with PI for 15 min at darkness, after washing with PBST for 15 min. Cell samples were observed using fluorescence microscope (Zeiss Axio Observer A1, Germany).

ROS levels were determined by staining cells with 10 μM CM-H₂DCFDA (Invitrogen, USA), and mitochondrial superoxide levels were determined by staining cells with 10 μM MitoSOX Red (Invitrogen, USA) following manufacturer's instruction. Mitochondrial morphology and membrane potential of cells were determined by staining cells with 100 nM MitoTracker Red-CMXRos (Life Technologies, USA) following manufacturer's instruction. Fluorescence-emitting dichlorofluorescein (DCF) were recorded using a fluorescence microscope (Axio Observer Z1). Mitochondria stained with MitoTracker Red-CMXRos were imaged with a LSM710 confocal microscope (Carl Zeiss, Germany). MitoSOX fluorescence was measured immediately using a flow cytometer (LSRII, BD Biosciences, USA).