Annexin 5 pi apoptosis detection kit

The Annexin-V/PI apoptosis detection kit (Becton Dickinson, San Diego, CA, USA) was used to evaluate the apoptosis of glioma cells induced by PTE according to the provided protocol. Briefly, T98G, LN18, LN229, and U87 glioma cells were treated with PTE (20, 40, and 80 µM) for 48 h. Next, collected cells were subjected to Annexin V-FITC/PI double-staining and then analyzed using a flow cytometer (Becton Dickinson, San Diego, CA, USA). The rate of cell apoptosis was analyzed using FlowJo version 10.0 software (FlowJo LLC/Becton Dickinson, Ashland, OR, USA).

Cells were transfected with control or miR-223-3p mimic. Apoptosis was assessed by measuring the membrane redistribution of phosphatidylserine using an Annexin V–PI apoptosis detection kit (Becton Dickinson).

MLO-Y4 cells were cocultured with different concentrations of CEFFE (50, 250, and 500 μg/ml), and cell cycle analysis was performed after 24 h. Cultured cells were collected and fixed with 70% ethanol overnight, followed by incubation with RNase A (Beyotime Biological Technology Institution, Shanghai, China) and propidium iodide (Beckman-Coulter, Brea, CA, United States).
Apoptosis of MLO-Y4 cells induced by Rosup (a compound mixture with 4-butylhydroperoxide included) was determined by flow cytometry using the Annexin-V/PI Apoptosis Detection Kit (Becton Dickinson and Co., Franklin Lakes, NJ, United States). In brief, MLO-Y4 cells were stimulated for apoptosis with Rosup, and the experimental group was incubated with CEFFE (250 μg/ml) for 8 h. In subsequent experiments to examine the mechanism of CEFFE anti-apoptosis, ERK, p38, and JNK inhibitors (10 μM), that is, GDC-0994, SB203580, and PD98059, were added to the cells incubated with Rosup and CEFFE for 8 h. Then the cells were harvested, washed twice with cold PBS, and labeled with FITC Annexin V and PI in a binding buffer. The cells were then submitted to flow cytometry using a BD LSR Fortessa system (Becton Dickinson and Co.) to detect the fluorescence intensity of the cells. The experiment was repeated three times, and the apoptosis rate (%) of each group was calculated.

Cells were treated with varying concentrations of anlotinib for the designated times. The cells were harvested and processed in accordance with the manufacturer's instructions. Propidium iodide (PI)/RNase staining buffer from BD Pharmingen (556463, New Jersey, USA) was used for cell cycle analysis. The cells were then examined using a NovoCyte flow cytometer with NovoExpress software (ACEA Biosciences, Inc., USA). An annexin V/PI apoptosis detection kit (BD Pharmingen, USA) was used to assess apoptosis. Cells positive for annexin V were determined to be apoptotic and were located in the dot plot's right quadrant. ANOVA was used for statistical analysis. P values less than 0.05 were considered significant when compared to the control group.

Cell proliferation and viability were determined by absorbance with MTT assay. Approximately 3000 cells per well were seeded in the 96‐well plates. At the indicated time‐points, 5 mg/mL MTT (Sigma) was added to the plates and incubated at 37°C for another 4 hours. Absorbance at 490 nm was measured with an automatic enzyme‐linked immunosorbent assay reader (BioTek Instruments). For cell apoptosis assay, cells were stained with Annexin V: PI Apoptosis Detection Kit (BD Bioscience) and processed using the BD FACSuite analysis software.