Annexin 5 fitc pi cell apoptosis detection kit

MCF-7 cells were seeded at approximately 120,000 cells/well in 6-well plates containing 2 mL of the medium. After 24 h, it was replaced with 2 mL of the fresh medium containing reagents at different concentrations. After a further 24 h incubation, the cells were washed twice with PBS, gently trypsinized, and pelleted by centrifugation at 500 × g for 5 min at 4 °C. They were resuspended in cold PBS, collected by centrifugation at 500 × g, and resuspended in 100 μL cold binding buffer. Each group was treated with 5 μL annexin V-FITC and 5 μL PI and incubated with fluorochrome for 15 min at room temperature. Before flow cytometry, a binding buffer (400 μL) was added to each group to detect apoptosis. The Annexin V-FITC/PI Cell Apoptosis Detection Kit was purchased from TransGen (Beijing, China), and the flow cytometer was obtained from Thermo Fisher Scientific (USA).
For the apoptosis assay of MCF-7 cells subjected to CDK7 downregulation or transfection treatment, the relevant genes were downregulated or upregulated before seeding. The following procedures were the same as those used for the drug-treated group.

The apoptosis of EL4 cells were determined by flow cytometry. Twenty-four hours after irradiation of 8 Gy, the EL4 cells were digested and stained using Annexin V-FITC/PI Cell Apoptosis Detection Kit (TransGen Biotech Corp., Ltd, Beijing, China) for 20 min, then flow cytometry was used to analyze apoptosis rate of EL4 cells.

Apoptosis was detected by flow cytometry using the Annexin V-FITC/PI cell apoptosis detection kit (Transgen biotech, Guangzhou, China). After stimulating the cells, they were washed twice in PBS before being resuspended in 100 μl of binding buffer and 5 μl of Annexin V-FITC; then, 5 μl of PI was added in dark conditions to stain the cell for 15 min according to the manufacturer's instructions.

The cells were collected by centrifuging for 5 min at the speed of 500 g, 4 °C. The cells were washed with pre-cooling PBS for 2 times. Cells were then resuspended in the Annexin V Binding buffer. The cell suspension was dyed with Annexin V-FITC and PI and plunged into darkness at room temperature for 15 min. Then, the cell suspension was mixed with Annexin V Binding buffer and put on ice. The apoptosis rate of cells was determined by flow cytometry in an hour. The assay was performed according to the instruction of Annexin V-FITC/PI Cell Apoptosis Detection Kit (TransGen Biotech, Beijing, China).

The apoptosis ratio was measured by AnnexinV-FITC/PI Cell Apoptosis Detection Kit (FA101-02, TransGen) according to the manufacturer’s instructions. Briefly, cells were trypsinized by non-EDTA trypsin and collected by centrifugation at 500 g, 4°C for 5 minutes. Then, cells were washed thrice with PBS and resuspended in 100 μL pre-chilled 1×Annexin V Binding Buffer, supplemented with 5 μL Annexin V-FITC and 5 μL PI. Cells were incubated at room temperature for 15 minutes in the dark. After incubation, 400 μL Annexin-binding buffer was added, and samples were immediately analyzed in CyAn ADP7 flow cytometer.

Flow cytometry was performed to detect the apoptosis of hippocampal neurones. Hippocampal neurones were seeded into 96-well plate and treated with Aβ_1-42 oligomer (10 μmol/L), IL-1β (10 μg/L) or AC-YVAD-CMK (10 μmol/L) for 24 h. After that, the cells were collected and washed with pre-cooled PBS for 2 times. Cells were then resuspended in the Annexin V Binding buffer. The cell suspension was dyed with Annexin V-FITC and PI and plunged into darkness at room temperature for 15 min. Then, the cell suspension was mixed with Annexin V Binding buffer and put on ice. The apoptosis rate of cells was determined by flow cytometry in an hour. The assay was performed according to the instruction of Annexin V-FITC/PI Cell Apoptosis Detection Kit (TransGen Biotech, Beijing, China).

Se-1 and Se-3 cells were seeded at a density of 2 × 10⁵ cells per well in 24-well plates for 24 h and then inoculated with WT-AcMNPV at an MOI of 5 for 1 h. After removing the viral inoculum, the cells were washed once with TNMFH (10% FBS) medium and then incubated. At different time points (1, 3, 6, 12, 24, 48 and 72 h) p.i., the infected cells were examined with an Annexin V-FITC/PI Cell Apoptosis Detection Kit (TransGen) and then observed and photographed under a fluorescence microscope (EVOS FL, Thermo Fisher Scientific, Waltham, MA, USA). The apoptotic process was divided into early apoptosis and late apoptosis. Cells in which only the membrane is stained green with FITC are in early apoptosis, while cells in which the nuclei are also stained red with PI are in late apoptosis. The apoptosis rates were calculated using randomly taken photos. The percentage of the total number of green and red cells to the total number of seeded cells was used as the apoptosis rate.