Annexin 5

Apoptosis of cells was detected by Annexin V staining (Yeason, China). After being extracted from the bone marrow of mice, 5 × 10⁶ cells were labeled with different surface markers for 30 to 45 min at 4 °C and then twice rinsed with PBS. Subsequently, the cells were reconstituted in binding buffer and supplemented with Annexin V. After 30 min of incubation, flow cytometry was detected in the FITC channel. Cell cycle analysis was performed with the fluorescein Ki-67 set (BD Pharmingen, USA), following the directions provided by the manufacturer. Briefly, a total of 5 × 10⁶ bone marrow cells were labeled with corresponding antibodies, as previously stated. Afterward, the cells were pre-treated with a fixation/permeabilization concentrate (Invitrogen, USA) at 4 °C overnight and subsequently rinsed with the binding buffer. The cells were stained with Ki-67 antibody for 1 h in the dark and then with DAPI (Invitrogen) for another 5 min at room temperature. Flow cytometry data were collected by a flow cytometer (CytoFLEX LX, Beckman, USA).

CAR‐NK cells were co‐cultured with target cells at different ratios in 24‐well plates with medium supplemented with 200 U ml⁻¹ IL2 for 24 h, and then cells were collected and stained with the CAR and apoptotic marker Annexin V (YEASEN) and then analyzed by flow cytometry.

Pericytes were co-stained with Annexin V and propidium iodide (Cat. #40305ES20; Yeasen). After three washes, the cells were assayed by flow cytometry, and the ratio of apoptotic or necrotic cells to normal control was calculated accordingly.

Cells (1.5 × 10⁵ cells/well) were seeded into 6-well plates and treated with Chidamide as described. They were collected and stained by Annexin V and propidium iodide (PI) (Yeasen, Shanghai, China) following a standardized protocol. Cell apoptosis was analyzed by flow cytometry (FACSCalibur; BD Biosciences, USA).