Mitosox red probe

hPDLSCs were detached by trypsin and recultured on glass-bottom culture dishes with a density of 5 × 104 cells per dish. When hPDLSCs were reached near 80%, the lipopolysaccharide (LPS) was added to the medium at 8 μg/L for 2 h, then 200 ng/ml MitoQ@PssL NPs were added in the medium and cultured for 24 h. The intracellular level of ROS was measured by using a DCFH-DA probe (Keygen, China). The mitochondrial level of superoxide was measured by using the MitoSOXTM Red probe (Yeasen, China). The fluorescence of DCF and MitoSOX were observed at 488 nm and 510 nm separately under a laser scanning confocol microscope (LSCM, A1R, Nikon, Japan) and FACS flow cytometry (BD Biosciences, United States). Using the ImageJ program (Media Cybernetics, United States), the quantitative study of fluorescence intensity was carried out.

BALB/c nude mice were purchased from Vital River Laboratories (Beijing, China), weighting about 20 g were chosen for the establishment of an oxidative stress model, LPS (0.02 ml, 1 mg/ml) daily subgingival injection administration. A total of 3 days later, MitoQ@PssL NPs were injected in situ with a dosage of 200 μl per site. A total of 24 h later, the MitoSOXTM Red probe (Yeasen, China) was injected to detect the remaining mtROS. The results of whole-body fluorescence imaging were used an IVIS^® Lumina Ⅲ imaging system (PerkinElmer, United States). ImageJ software was used to measure and quantify the fluorescence signal intensity.

After the GCs were treated as described in the study, mitochondrial ROS (mtROS) levels in each group were assessed using the MitoSOX Red probe from Yeasen (Shanghai, China), following the manufacturer’s instructions. The cells were incubated at 37 °C in the dark for 10 min, followed by three washes with PBS. Next, 50 nM MitoTracker™ Green FM (ThermoFisher, Waltham, MA, USA, M7514) was added, and the cells were further incubated at 37 °C in the dark for 30 min. After three additional washes with PBS, ROS levels in the GCs were visualized and captured using a laser-scanning confocal microscope (LSM700ME-TA; Zeiss, Oberkochen, Germany). Subsequently, the obtained results were quantified and analyzed for each group using ImageJ 1.42q software.