Annexin 5 fitc pi apoptosis assay kit

MDA-MB-231 cell apoptosis was analyzed using an Annexin V-FITC/PI apoptosis assay kit (Neobioscience, China). Briefly, cells grown in the DECM scaffolds were harvested with trypsin. Cells were then resuspended in binding buffer and stained with Annexin V-FITC and PI for 15 min at room temperature in the dark. For cell cycle analysis, MDA-MB-231 cells prepared from either 3D cultures (at Day 10) or 2D cultures were treated with cold 75% ethanol at 4 °C overnight. Fixed cells were washed with cooled PBS and incubated with propidium iodide solution (Keygen, China) for 30 min at room temperature. All analyses were performed using a FACS Calibur analyzer (BD Biosciences, USA) with FlowJo software (Tree Star Software, San Carlos, California, USA).

Breast cancer cells SK-BR-3 and MDA-MB-231 cells were cultured in suspension or adherent condition for 3 d. Cells were collected and re-suspended. For cell proliferation assays, 5×10³ cells were seeded in 96-well plates with 10% FBS. After 3 h, 24 h, 48 h and 72 h, 100 μL new medium with 10 μL MTS (G5421, Promega, Madison, USA) was added into each well. The absorbance at 490 nm was measured. For cell survival assays under nutrient deficiency condition, 1×10⁴ cells were seeded in 96-well plates without FBS and cultured for 4 d. The cell activity was detected using MTS assay. For cell cycle profiling, Cell Cycle Analysis Kit (C1052, Beyotime, Beijing, Shanghai, China) was used according to the manufacturer's protocol. In brief, cells were collected and fixed overnight with 70% ethanol at 4 ºC. Cells were suspended and incubated in PBS containing propidium iodide (PI) and RNAase at 37 ºC for 30 min. Then, the stained cells were analyzed by BD FACS Calibur Flow Cytometer. For apoptosis assays, cells were stained with Annexin V and PI using the Annexin V-FITC/PI apoptosis assay kit (FAK015.60, Neobioscience, Shenzhen, China), then analyzed by flow cytometry.

We used an annexin V-FITC/PI apoptosis assay kit (NeoBioscience, China) to detect cellular apoptosis. After digestion with trypsin, the cells were stained with Annexin V-FITC for 10 min. Then, PI was added to further stain the cells in the dark for 5 min. An AccuriC6 flow cytometer (BD, USA) was used to analyze the cells according to the manufacturer’s instructions.

Hepatocellular carcinoma cells were cultured for 48 h after EA (7, 14 and 28 µM) and DMSO treatment. The cells were then collected, washed with cold PBS and suspended in 200 µl binding buffer (Annexin V-FITC/PI apoptosis assay kit; Neobioscience Technology Co., Ltd.) at a density of 5×10⁵/ml. Subsequently, cells in binding buffer (195 µl) were incubated with 5 µl Annexin V-FITC at room temperature in the dark for 10 min. The cells were then washed with PBS, suspended in binding buffer, and mixed with 10 µl PI. The mixture was gently mixed and analyzed by DxFLEX flow cytometry. The apoptotic rate of the hepatocellular carcinoma cells was finally determined using CytExpert 2.0 software (Beckman Coulter, Inc.).