Dcfh da

The ROS production in different samples was determined by 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) (YEASEN, Shanghai, China). Briefly, HK-2 cells were seeded in the 6-well plates at 2 × 10⁵ cells/well and pretreated with 0, 1, 10 and 50 µM zoledronate at 37 °C for 6 h, and then 500 µl DCFH-DA (10 µM final concentration) was added to each well incubated at 37 °C for 30 min. After washing three times with PBS, cells were harvested by cell scrapers and resuspended in PBS, were analyzed immediately using flow cytometry (Ex = 488 nm, Em = 525 nm).

MRSA suspensions (10⁸ CFU/mL) were added into 6-well plates and treated with blank LB, EM, M-LFLIU, or EM+M-LFLIU (EM, 6.25 μg/mL, and M-LFLIU, 1 W/cm²). After treatment, the suspensions were incubated at 37°C for 3 h, and the bacterial cells were collected, stained with DCFH-DA (2′,7′-dichlorodihydrofluorescein diacetate; Yeasen, Shanghai, China) at 37°C for 30 min, and imaged under a confocal microscope. The ROS-positive rates for each sample were measured by a CytoFlex S flow cytometer (Beckman Coulter, USA).

MRSA suspensions (10⁸ CFU/mL) were added into 6-well plates and treated with blank LB, EM, M-LFLIU, or EM+M-LFLIU (EM, 6.25 μg/mL, and M-LFLIU, 1 W/cm²). After treatment, the suspensions were incubated at 37°C for 3 h, and the bacterial cells were collected, stained with DCFH-DA (2′,7′-dichlorodihydrofluorescein diacetate; Yeasen, Shanghai, China) at 37°C for 30 min, and imaged under a confocal microscope. The ROS-positive rates for each sample were measured by a CytoFlex S flow cytometer (Beckman Coulter, USA).

After anesthesia, mice were intracardially perfused with ice-cold PBS to remove the blood. Next, the L3–L5 spinal cord was dissected to prepare a single-cell suspension. The isolated cells were diluted to 1 × 10⁷ cells/mL and seeded into a 24-well plate. The cells were then incubated with DCFH-DA (10 μM, 50101ES01, Yeasen Biotechnology, China) for 30 min at 37 °C. After washing the cells thrice with serum-free medium, they were suspended in PBS and analyzed by flow cytometry.

Measurement of intracellular ROS was performed using 2,7-dichlorofluorescin-diacetate (DCFH-DA) (Yeasen, Shanghai, China). Cells were incubated with 10 μM DCFH-DA diluted in RPMI-1640 medium for 30 min at 37 °C in the dark. The cells were then immediately observed under the fluorescence microscope. Fluorescence intensity was quantified using ImageJ software. For flow cytometry, cells were collected for analysis. For tissue ROS measurement, frozen tumour sections were incubated with DCFH-DA (Item No: E004, Jiancheng Bioengineering, Nanjing, China) at 37 °C for 30 min. The ROS level in the tissue was measured under the BX41 fluorescence microscope.

UDCA (MCE, cat: HY-13771); fetal bovine serum (Biological Industries); trypsin (Beyotime, cat: C0201), penicillin–streptomycin (Beyotime, cat: C0222), reactive oxygen fluorescent probe (DCFH-DA, Yeasen, cat: 50101ES01); reverse transcription kit (Vazyme; code: R333-01); DMEM medium (Gibco), hydrogen peroxide (H₂O₂, Sigma, CAS-No: 7722-84-1); SYBR Green Mix (Vazyme); CCK-8 kit (Beyotime, cat: C0037); H₂O₂ kit (Solarbio, cat: BC353); and H₂O₂ kit (Solarbio, cat: BC3595); MDA kit (Beyotime; cat: S0131S); SOD kit (Beyotime; cat: S0101S); ROS kit (Yeasen; cat: 50101ES01).
Enzyme Labeling Instrument (BioTek, cat: Cytation3); Fluorescence Quantitative PCR Instrument (Applied Biosystems, USA, ABI PRISM®7900HT system).

Lipofectamine 2000, Dulbecco's modified Eagle's medium (DMEM), α‐minimal essential medium (α‐MEM), Opti‐MEM, fetal bovine serum, horse serum, penicillin‐streptomycin, 0.25% trypsin‐EDTA, DiO dye, Exosome Spin Columns, Micro BCA Protein Assay kit, as well as CD11b, CD86, CD206, CD11c, CD80 monoclonal antibodies were purchased from Thermo Fisher Scientific, USA. Calnexin, CD9, CD63 and CD81, FASL, Caspase‐3, Caspase‐8, and PARP antibodies were purchased from Abcam. 3‐(4, 5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT), RNAlater, dimethyl sulfoxide (DMSO), LysoTracker Green DND‐26, isothiocyanate‐labeled phalloidin and Hoechst 33342 were purchased from Sigma Aldrich, USA. Filter membranes and ultrafilter tubes were purchased from Millipore (Shanghai, China). Cy5‐labeled siRNA (Cy5‐NC), siPLK1 (targeting PLK1), and siPD‐L1 (targeting PD‐L1) were supplied by Suzhou Ribo Life Science Co., Ltd. (Suzhou, China) or Suzhou Biosyntech Co., Ltd (Suzhou, China). All the primers were provided by BioSune Co., Ltd (Shanghai, China). DCFH‐DA, Annexin V‐FITC/PI Apoptosis Detection Kit, and Hieff qPCR SYBR Green Master Mix were purchased from Yeasen (Shanghai, China). Chlorin e6 (Ce6) was purchased from Macklin Biochemical Technology Co., Ltd (Shanghai, China). Optimal cutting temperature (OCT) compound was from Sakura Finetek USA, Inc. (Torracne, CA90501, USA).