Jc 1 probe

Cells were digested with 0.25% trypsin and incubated with JC-1 probes (Beyotime, Shanghai, China) at 37°C for 20 min. After being washed with JC-1 1 × buffer twice, the cells were suspended in 500 μL buffer. The green fluorescence of JC-1 monomers (an indicator of a low MMP) and red fluorescence of aggregated JC-1 (an indicator of a high MMP) were captured by using a flow cytometer (BD LSRFortessa™; BD Biosciences, San Jose, CA, USA). In addition, the relative ratios of green fluorescence/red fluorescence were calculated to evaluate the mitochondrial membrane potential in cells.

The levels of superoxide ions and the mitochondrial membrane potential (MMP) in treated BMSCs were determined by MitoSox and JC-1 probes, respectively, according to the manufacturer's instructions (Beyotime, Shanghai, China). The cells were observed using a fluorescent microscope (Olympus Life Science; Tokyo, Japan).

Mitochondrial membrane potential (MMP) was detected using JC-1 probes (Beyotime). The monomeric form of JC-1 emits green fluorescence at a low MMP, while the aggregate form emits red fluorescence at a higher MMP. After the corresponding treatments, a JC-1 dye solution was added to the cells for 20 min at 37°C, followed by the JC-1 dye buffer twice. Next, inverted fluorescence microscopy (Leica) was used to capture the images. The MMP was represented by the ratio of red to green fluorescence.

The MMP was identified via JC-1 probe (Beyotime Biological Technology, China). A549 cells were pretreated with AC for 2 h and then exposed to RSV for another 24 h. After that, the cells were cleaned in PBS and incubated with JC-1 work solution (10 μM) under 37°C without light for 20 min. The cellular staining was identified via flow cytometry.

Mitochondrial membrane potential (MMP) was detected using JC‐1 probe (Beyotime, China). Macrophages were seeded in a 96‐well half‐area microplate (Greiner) (4.5 × 10⁴ cells/well) and treated as described above. After washed with PBS, cells were incubated with JC‐1 staining solution for 20 min followed by washing with JC‐1 staining buffer for two times. Fluorescence intensity was obtained by a Fluorescence microplate reader with excitation/emission at 490/530 nm (monomers) or at 525/590 nm (aggregates).

Mitochondrial were visualized with the MitoTracker Red CMXRos dye (Yeasen, China). Briefly, primary GCs were were plated on coverslips and incubated with 200 nM MitoTracker Red CMXRos at 37 °C for 30 min. Mitochondrial membrane potential was determined by the JC-1 probe (Beyotime, China) according to the supplier’s instructions. The red and green JC-1 fluorescence ratio was calculated. Images were quantified with Image-Pro Plus 6.0.

HUVECs (8×10⁴ cells/ml) were seeded in 6-well plates for 24 h and treated as required. Cytosolic Ca²⁺, MMP, cell cycle parameters, and apoptosis were respectively detected by the Fluo-4 AM probe (Beyotime Biotechnology, Shanghai, China), the JC-1 probe (Beyotime Biotechnology, Shanghai, China), PI/RNase Staining Buffer (BD Biosciences, CA, United States), and Annexin V-FITC/PI apoptosis detection kit (BD Biosciences, CA, United States), according to the manufacturers’ instructions, using a flow cytometer (BD C6 Plus, BD Biosciences, CA, United States). The data were analyzed using FlowJo 10.6 software (BD Biosciences, CA, United States). A CLSM (Carl Zeiss AG, BW, Germany) was used to capture the fluorescence images of MMP (Qin et al., 2021 (link); Yu et al., 2021 (link); Wu et al., 2022 (link)).