Annexin 5 pe 7 aad kit

The human tongue squamous cell carcinoma cell SCC-9 was purchased from Shanghai Yaji Biotechnology Co., Ltd.; fetal bovine serum (FBS) from Shanghai Ikesai Biological Products Co., Ltd.; CCK-8 cell proliferation/toxicity detection kit from Beijing Quanshijin Biotechnology Co., Ltd.; Annexin V PE/7AAD kit and PI/RNase staining buffer from Becton, Dickinson and Company in the United States; human FGF-3 Protein from R&D Systems in the United States, PD173074 (FGFR inhibitor) from MedChemExpress in the United States; and both primary and secondary antibodies from Abcam in the United States.

The apoptotic rates of U87 and U251 cells were detected using an Annexin V-PE/7-AAD kit (Becton Dickinson, New Jersey, United States). Cells were seeded in 6-well plates and treated with 0, 0.5, 2 or 8 µM GNE-477 for 48 h. After digestion with EDTA-free trypsin, cells were collected by centrifugation at 4°C. Subsequently, the cells were washed twice with PBS precooled at 4°C, 100 µL of 1x binding buffer were added to resuspend the cells, and the density was adjusted to 1 × 10⁶ cells/ml. Then, 5 µL of Annexin V/PE and 10 µL of 7-AAD were added, mixed gently and incubated with the cells at room temperature for 15 min in the dark. Then, 400 µL of 1 × Binding Buffer were added to each tube. Finally, apoptotic cells were detected using a FACS Calibur flow cytometer (BD Biosciences, United States), and the results were statistically analyzed using FlowJo software (Tree Star, Ashland, OR, United States).

Annexin V-PE/7-AAD kit (Becton Dickinson, USA) were used to measure the apoptosis of glioma cells. As request of the manufacturer's instruction, Cells were digested and collected, washed for 3 times, stained with Annexin VPE/7-AAD for 10 min in the dark. Apoptosis was analyzed by FACS Calibur flow cytometer (Becton Dickinson). PE Annexin V and 7-AAD negative indicate viable; PE Annexin V positive and 7-AAD negative indicate early apoptosis; PE Annexin V and 7-AAD positive indicate late apoptosis or dead. Early apoptosis and late apoptosis were summed and the total apoptosis rate was calculated.

Apoptosis was assessed by flow cytometry using a BD Accuri C6 (BD Biosciences). The Annexin V-PE/7AAD Kit (BD Bioscience) was used for analysing apoptosis and performed in accordance with manufacturer's instructions. Data were analysed using the FlowJo V7 software (Tree Star, OR, USA). Experiments were performed independently in triplicate for this study.