Annexin 5 fitc pi double staining apoptosis detection kit

Cells were first transfected with plasmids using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s instructions. Briefly, cells (1 × 10⁵) were seeded into 24-well plates overnight before transfection. Cells were then transfected with 500 ng of various plasmids including pDCUg-NT, pDCUg-F, pDCUg-L, pDCUg-FL, pDM-NT, pDM-F, pDM-L, and pDM-FL. The mouse and human cells were transfected with vectors targeting to mouse and human genes, respectively. The transfected cells were cultured for 24 h and then incubated with or without 50 μg/mL of FeNP, and cells were cultured for another 72 h. For HL7702 and MRC5, cells were first induced with or without 10 ng/mL TNF-α (Sigma) for 1 h before FeNP treatment. At 24 h, 48 h, and 72 h post FeNP administration, all cells were stained with AO&EB following the manufacturer’s instructions. Cells were imaged under a fluorescence microscope (IX51, Olympus) to observe numbers of live and dead cells, and living cell numbers were counted from the cell images by the Image-Pro Plus software. To quantify cell death, cells were collected at 72 h post FeNP administration and detected with the Annexin V-FITC/PI double staining Apoptosis Detection Kit (BD, USA) according to the manufacturer’s instructions. The fluorescence intensity of cells was quantified with CytoFLEX LX Flow Cytometer (Beckman) and CytExpert software.

Flow cytometry was used to analyze cell apoptosis. In this experiment, ~60% confluent cells were seeded into six-well plates and treated with CuD or gefitinib for 72 h. The apoptosis assay was performed with an Annexin V-FITC/PI double staining apoptosis detection kit (BD Biosciences) and a BD FACSCalibur Flow Cytometer following the manufacturer's instructions.