Mitosox

MitoSOX (Yeasen, Cat#40778ES50) and DCFDA/H2DCFDA dye (Abcam, Cat#ab113851) were used for the determination of mitochondrial and cellular reactive oxygen species. TMRM (Abcam, Cat#275547) was used for the determination of membrane potential. The corresponding channels were detected by flow cytometry.

Levels of intracellular ROS and mitochondrial ROS (mtROS) generation were detected using 2’,7’- dichlorodihydrofluorescein diacetate (DCFH-DA) and MitoSOX (Yeasen, China). The prepared tissue sections were treated with 10 M DCFH-DA for 20 min at 37 °C in the dark, humid chamber. The cell slides were treated with 2 mL MitoSOX red mitochondrial superoxide indicator for 10 min at 37 °C in the dark, humid chamber. The ROS positive counts from four randomly chosen fields observed under a fluorescence microscope were measured.

Fluorescent dye 2′,7′‐dichlorofluorescin diacetate (H2DCF‐DA; Beyotime) were used to measure the intracellular ROS according to the manufacturer's instructions. Mitochondrial ROS were examined by MitoSOX (YEASEN) according to the manufacturer's instructions.

H9C2 cells were placed on poly‐L‐lysine‐treated coverslips in 24‐well plates and incubated with 1 ml of DMEM with 10% FBS. After the cells had grown to the appropriate density, they were stimulated with 400 µM H₂O₂ or 100 µM erastin for 6 h with or without PBS or SSSe NPs (10 ng ml⁻¹). After the culture medium was removed at the end of the treatment period, the cells were stained with 5 µM MitoSOX (Yeasen, Shanghai, China) for 10 min at 37 °C in an incubator in the dark. Then, the cells were washed 3 times with 37 °C PBS and observed under a fluorescence microscope (Leica DM3000 LED).

Mitochondrial ROS and MMP were measured by Mito-SOX (40778ES50; Yeasen biotech, Shanghai, China) and Mitotracker (C1048, Beyotime). The NPCs were seeded in the logarithmic growth period on a 6-well plate, cultured at 37 °C overnight under 5% CO2 saturated humidity and treated as the experimental design. The NPCs were stained and fixed according to the manufacturer’s instructions. Finally, the Mito-SOX and the Mitotracker intensity were observed using the LSM.

Cells were washed and dissociated using trypsin without EDTA (Beyotime, #C0207). Induction of apoptosis was determined with the Annexin V, FITC Apoptosis Detection Kit (DOJINDO, #AD10) according to manufacturer’s instructions. Loss of mitochondrial membrane potential was evaluated with TMRM (100 mM, Thermo Fisher, #T668). Mitochondrial ROS were detected with MitoSOX (5 µM, Yeasen, #40778ES50). Endocytosis of DiO-labeled OMVs after pre-treatment of cells with chemical inhibitors was determined 2 h after OMV challenge. All samples were run on a DxFLEX (Beckman) following the gating strategy in Supplementary Fig. 8 to select for single cells and Flow Jo X 10.0.7 software was used for data analysis.

HeLa cells (2 × 10⁵ cells/dish) were seeded in glass bottom cell culture dishes (NEST, #801002) and transfected with expression vectors for (fluorescent)-tagged proteins or used in OMV endocytosis or gentamicin protection assays. For identification of endosomes, Cascade Blue-conjugated dextran 10,000 MW (Thermo Fisher, #D1976) was added to cell culture media (5 mg/mL). For specific fluorescent labeling of mitochondria, MitoBright LT Red (DOJINDO, #MT11) was added to the cell culture medium (0.1 μM). For evaluation of the mitochondrial membrane potential, TMRM (Thermo Fisher, #T668) was added to the cell culture medium (100 mM). For detection of mitochondrial ROS, MitoSOX (Yeasen, #40778ES50) was added to the cell culture medium (5 µM). Images were acquired on Zeiss LSM880 or Olympus FV3000 microscopes while maintaining the cells in a heated CO₂ incubator. High-resolution images acquired with the Zeiss LSM880 microscope were used for 3D reconstruction with Imaris 9.5 software.