Apoptosis and necrosis detection kit

The viability and apoptosis of ECs and C2C12 cells were detected as described in our previous study [26 (link)]. In brief, cell viability was detected by a CCK-8 assay kit (Beyotime, China) based on the manufacturer’s instructions. Cell apoptosis was measured by using an Annexin V-PE/7-AAD apoptosis detection kit (BD bioscience, USA) followed by flow cytometric analysis. EC necrosis was measured by using an Apoptosis and Necrosis Detection Kit (Beyotime, China) based on the manufacturer’s instructions, followed by immunofluorescence analysis. After coculture with EXs, ECs were incubated with 1 ml of YPI/PI working solution at 37 °C for 20 min and then observed under a fluorescence microscope. The cells that were double positive for YO-PRO-1 and PI staining were considered necrotic ECs.

A total of 5 × 10⁴ cells within 0.1 mL complete medium per well were seeded into 96-well plates. When the logarithmic stage was reached, 0.1 μg/well plasmid or shRNA was transfected into cells for 36 h. Then cells were challenged with S. aureus solution for 6 h at an MOI of 10:1. Afterward, the Apoptosis and Necrosis Detection Kit (Beyotime, Shanghai, China) was used to determine cell apoptosis and necrosis. All determination steps were performed following the manufacturer’s instructions. Finally, the fluorescence value was determined using the SpectraMax i3x Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA, USA).
For fluorescence staining, a total of 2.5 × 10⁵ cells within 0.5 mL of complete medium per well were seeded into 24-well plates. Similarly, after transfection and S. aureus challenge, cell apoptosis was determined by the above Apoptosis and Necrosis Detection Kit. Samples were photographed by Nikon ECLIPSE Ts2 fluorescence microscope (Nikon, Tokyo, Japan). Apoptotic cells were stained green by YO-PRO-1. In this section, Mac-T cells were challenged with the representative S. aureus strain Newman.

Radioimmunoprecipitation assay (RIPA) lysis buffer, bicinchoninic acid assay (BCA assay), protein loading buffer, bovine serum albumin (BSA), protease inhibitor cocktail, coomassile blue, cell count kit‐8 (CCK8) assay, 4% paraformaldehyde, triton‐X100, apoptosis and necrosis detection kit, and EdU cell proliferation kit were purchased from Shanghai Beyotime Biotechnology. Phosphate‐buffered saline (PBS), Dulbecco's modified Eagle's medium (DMEM), 0.25% trypsin‐EDTA, Roswell Park Memorial institute‐1640 (RPMI‐1640), fetal bovine serum (FBS), and penicillin‐streptomycin (10 000 U mL⁻¹) were purchased from Thermo Fisher Scientific, Inc. The live/dead fluorescence assay reagent, PKH67 dye, and lipopolysaccharide (LPS) were purchased from Sigma‐Aldrich. The catalase (CAT) and superoxide dismutase (SOD) activity detection kits were obtained from Nanjing Jiancheng Co., Ltd. The 4′,6‐diamidino‐2‐phenylindole (DAPI), 2′,7′‐dichlorofluorescin diacetate (DCFH‐DA) and Hoechst 33 342 were purchased from Beijing Solarbio Life Science Inc.