Annexin 5 pe 7 aad staining kit

Cell death was evaluated by using an Annexin V-PE/7-AAD staining Kit (BD Biosciences, San Jose, CA). Cultures were rinsed with ice-cold PBS and incubated with accutase (Life Technologies, Carlsbad, CA) at 37 °C for 2 min. The detached cells (from culture medium, PBS wash, and accutase treatment) were collected by centrifugation (2000 rpm, 5 min). Cell pellets were further washed twice with ice-cold PBS and resuspended in 100 μl 1× binding buffer from the Annexin V-PE/7-AAD staining Kit. 5 μl of PE Annexin V and 5 μl of 7-AAD were added to the cells and incubated at room temperature for 15 min. After incubation, 400 μl binding buffer was added to each sample. Cells were analyzed using a Becton Dickinson Biosciences FACSCalibur (BD Biosciences, San Jose, CA).

For flow cytometry analysis of annexin V externalization, cells were seeded on a 6-well plate (2 × 10⁵ cells per well) to achieve 70% confluence overnight before assay. AGS cells were transfected with Cav-1 overexpression vector or empty vector for 24 h, followed by incubation with 20 μg/ml cisplatin for 12 and 24 h, respectively. MGC803 cells were transfected with shCav-1 vector or negative control vector for 48 h, followed by incubation with 8 μg/ml cisplatin for 12 and 24 h, respectively. Annexin V levels were determined using an Annexin V-PE/7-AAD staining kit (BD Pharmingen, San Diego, CA, USA) according to the manufacturer's protocol. All cells were analyzed by flow cytometry using a BD FACSCanto II flow cytometry (BD Pharmingen) to detect the fluorescence signal.

Cells were digested and resuspended in D'PBS after transfection. Annexin V‐PE/7‐AAD staining kit (BD Biosciences, San Jose, CA, USA) was used for cell staining according to the manufacturer's instructions. The apoptosis rates of different groups were detected by FACS after 15‐min staining. Three independent experiments were performed.

MH7A cells were seeded in 6-well plates and incubated with different concentrations of WTD (1 or 10 mg/ml) for 24 h. Adherent MH7A cells were digested with trypsin without EDTA. The cells were resuspended in 200 μL of 1× binding buffer in a centrifuge tube, and the concentration of cells was adjusted to 1×10⁶ cells/ml. Then, apoptosis was detected with an Annexin V-PE/7-AAD staining kit (BD Biosciences) and assayed by flow cytometry (BD FACS caliber; BD Biosciences). Data analysis was conducted using FlowJo software.

Cell death was evaluated by using an Annexin V-PE/7-AAD staining Kit (BD Biosciences, San Jose, CA). Cultures were rinsed with ice-cold PBS and incubated with accutase (Life Technologies, Carlsbad, CA) at 37 °C for 2 min. The detached cells were collected by centrifugation (2000 rpm, 5 min). Cell pellets were further washed twice with ice-cold PBS and resuspended in 100 μl 1× binding buffer from the Annexin V-PE/7-AAD staining Kit. Five microlitre of Annexin V-PE and 5 μl of 7-AAD were added to the cells and incubated at room temperature for 15 min. After incubation, 400 μl binding buffer was added to each sample. Cells were analyzed using a Becton Dickinson Biosciences FACSCalibur (BD Biosciences, San Jose, CA).