Annexin 5 pi double staining kit

For cell cycle analysis, cells near 50% confluence were synchronized in the G₀/G₁ phase by overnight incubation in serum-free medium. Cells were then incubated in the complete medium containing 25 nM or 50 nM OTSSP167. After 24 hours (BGC823) or 18 hours (SGC7901) of incubation, the cells were trypsinized, washed with PBS, and fixed with 70% ethanol for 16 hours at −20°C. The samples were washed with PBS and stained with PI/RNase Staining Buffer (BD Biosciences) for 15 minutes. Cell cycle analysis was performed by fluorescence flow cytometry on a FACScan machine (BD Biosciences). For apoptotic analysis, cells were stained using an Annexin V/PI double staining kit (DOJINDO, Japan) according to the manufacturer's protocol.

The cellular apoptosis rate was determined using the Annexin V/PI double-staining Kit (Dojindo Laboratories, AD10). Cells were seeded in 6-well culture plates at a density of 2.0 × 10⁵ cells/well. After incubation with different concentrations of l-sorbose for 24 h, the cells were washed twice with annexin V binding buffer and double-stained with annexin V and PI. Cell apoptosis was examined on a FACSCalibur flow cytometer (BD Accuri C6) and analyzed by FlowJo software.

Cardiomyocytes were plated into 24-well plates at a density of 6.0 × 10⁴ cells/well and treated according to the experimental design. The apoptosis of cardiomyocytes was analyzed by flow cytometry using Annexin V/PI double staining kit (Dojindo) strictly according to the manufacturer's introduction. Cell apoptosis was measured by flow cytometry (Beckman, CytExpert).