C1031

Target YAC‐1 cells were labelled with carboxyfluorescein succinimidyl ester (CFSE; C1031, Beyotime) at a final concentration of 5 μM. They were then co‐cultured with effector NK cells at an effector‐to‐target cell (E:T) ratio of 2:1 or 0:1 for 4 h at 37°C under 5% CO₂. After 4 h, the samples were stained with Helix NP NIR (425301, BioLegend) and Annexin V‐PE (640908, BioLegend) for 10 min. Fluorescence‐activated cell sorting (FACS) analysis was performed immediately with a BD LSR Fortessa Cell Analyzer (BD Biosciences). The Incucyte S3 Live‐Cell Analysis Instrument (Sartorius AG) was used to monitor the killing activity of NK cells. Propidium iodide (C1052, Beyotime) was used to mark dead cells.

To evaluate the transfer of human platelet-derived mitochondria to mouse BA-MSCs, BA-MSCs were seeded in 12-well glass slides with 2 × 10⁵ cells per well. The cells were cultured overnight until all cells adhered to the wall. MitoTracker Red CMXRos labeled mitochondria were cocultured with BA-MSCs. At 4 h and 8 h, the transfer of mitochondria into BA-MSCs was detected using Pannoramic MIDI II scanner (3DHISTECH, Hungary). Before detection, the cytoplasm of BA-MSCs was labeled with carboxyfluorescein diacetate and succinimidyl ester (CFSE) (#C1031, Beyotime), and the nucleus of BA-MSCs was labeled with DAPI (#C1002, Beyotime).