Apoptosis Detection in TIB-152 Cells

Cell apoptosis was detected by an Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit (Nanjing KeyGen Biotech Co., Ltd.) according to the manufacturer's instructions. In brief, TIB-152 cells were seeded in 6-well plates (2×10⁵ cells/well) with RPMI-1640 supplemented with 10% FBS. Tan IIA (20 µM), IM (5 µM) or IM (5 µM) + Tan (20 µM) was added and the cells were cultured for 24 h. The TIB-152 group was treated with 0.1% DMSO. For rescue experiments, TBI-512 cells were pre-treated with IGF-1 (10 nM) 24 h before exposure to Tan IIA or/and IM. After 24 h of incubation, the cells were collected, centrifuged at 1,000 × g for 5 min and then resuspended in 500 µl binding buffer (provided with kit), followed by the addition of Annexin V-FITC (5 µl) and PI (5 µl). The samples were then incubated in the dark at room temperature for 15 min. Cell apoptosis assay was performed within 1 h on a flow cytometer (FACSCalibur system) equipped with Cell Quest software (version 5.1; BD Biosciences). The total percentage of apoptotic cells was defined as the sum of early and late apoptotic cells (22 (link)).