Annexin 5 propidium iodide kit

The induction of A549 resistant cells was performed by gradually increasing the concentration of cisplatin from a starting concentration of 1.0 µM, and after 4 weeks, it was increased to 2.0 µM, and serially subcultured for ~100 passages. Then, 6 months later, the A549/DDP resistant cell line was established. The human lung adenocarcinoma cells A549 were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China; cat. no. CL-0016). The cells were maintained in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) medium with 10% fetal bovine serum (FBS; Hyclone; GE Healthcare Life Sciences) and 1% penicillin-streptomycin solution (Beyotime Institute of Biotechnology, Haimen, China), and cells were cultured in a humidified atmosphere at 37°C with 5% CO₂. Co-culture systems of A549/DDP plus DC or DC-T were established. OMT (1 mg/ml)-treated DCs and the FOXP3⁺/CD₄⁺ T cells induced by DC-T co-culture (cell ratio, 1:5) were incubated with A549/DDP cells. The cells were seeded for 24 h in the upper and lower chambers of Transwell plates at ratios of 1:1, 1:5, 1:10 and 1:20. At the same time, 22 µM DDP was added to the A549/DDP medium for 24 h. The collected cells were stained using an Annexin V/Propidium Iodide kit (BD Biosciences) according to the manufacturer's protocol, and the apoptotic ratio was analyzed using a flow cytometer (BD Accuri C6; BD Biosciences).

Cells (3 × 10⁵/well) were plated in 6-well plates and transfected with siRNA vector or siKMT2D. Cell cycle distribution was analyzed with PI staining (BD Biosciences, Auckland, New Zealand); For cell apoptotic assay, cells were transfected for 48 h and assessed by Annexin V- Propidium Iodide kit (BD Biosciences, Auckland, New Zealand). The stained cells were acquired by flow cytometry (BD Biosciences, San Diego, CA, USA) and analyzed by FlowJo v7.6 software.