Annexin 5 fitc kit

An Annexin V-FITC Kit (Beyotime, C1062) was used to detect the apoptotic cells. For determining mitochondrial membrane potential (MMP) and analyzing ROS production, cells were stained using the TMRE Mitochondrial Membrane Potential Assay Kit (Abcam, Cambridge, UK, ab113852) and MitoTracker^® Red CMXRos (Yeasen, 40741ES50) and analyzed by flow cytometry (FACSCanto, BD) within 1 h.

H9c2 cells were seeded in black 96-well cell culture plates. Twenty-four hours after seeding, cells were incubated with DOX and ISL for 24 h. Then, apoptosis/necrosis assays were performed using the Annexin V-FITC kit (Beyotime). A mixture was prepared by mixing Annexin V-FITC conjugate and Annexin V-FITC solution at a ratio of 39:1, and the mixture was then supplemented with 10 μg/mL Hoechst 33,342 solution to get the working solution. For staining, the plates were centrifuged at 1000 RCF for 5 min, and then the cells were washed once with PBS. After aspirating the PBS, staining working solution was added to each well in a light-protected environment and incubated at room temperature for 15 min, followed by immediate image acquisition on the ImageXpress Micro Confocal imaging system using a 20× objective, with DAPI channel and FITC channel selected. The percentage of Annexin V-FITC-positive (apoptotic/necrotic) cells was analyzed using the Cell Scoring module in MetaXpress software.

Cell cycle and apoptosis analyses were performed as described previously [25 (link), 26 (link)]. EC9706 (1 × 10⁴/well) were seeded in 96-well plates and incubated overnight, and treated with different doses of curcumin (0, 4, 8, 12, 16 μM) for 24 h. For apoptosis detection, the cells were stained with Hoechst (Beyotime, Shanghai, China) and Annexin V–FITC Kit (Beyotime, Shanghai, China). For cell cycle analysis, the curcumin-treated cells were fixed with 4% paraformaldehyde and stained with DAPI (Solarbio, Beijing, China) according to the protocol and protected from the light for 20 min at 37 °C in a 5% CO2 incubator. They were analyzed by In CELL Analyzer 2000.

Osteosarcoma cell MG-63 and U2-OS were purchased from Shanghai Cell Bank of Chinese Academy of Sciences, and osteosarcoma tissue before and after radiotherapy was obtained from local Cancer Hospital. Fetal bovine serum and DMEM (dulbecco’s modified eagle medium) culture were purchased from Gibco, USA; RNA extraction kit, reverse transcription kit and qRT-PCR (Quantitative Real-time Polymerase Chain Reaction) kit were purchased from Takara, Japan; Lipofectamine^TM 2000 transfection kit was purchased from Invitrogen, USA; MTT (3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide) kit, Annexin V-FITC kit and propidium iodide (PI) kit, dual luciferase reporter gene assay kit were purchased from Beyotime Biotech Inc., Shanghai; Dimethyl sulfoxide (DMSO), BCA kit, RIPA protein lysate, and SDS-PAGE kit were purchased from Sigma; Co60 medical irradiation device was purchased from Nuclear Power Institute of China.