Fitc conjugated annexin 5 apoptosis detection kit

Cell viability was assessed using the calorimetric WST assay, direct cell counting, and Annexin V staining. For the WST assay, HeXS34 cells or BMMCs treated with the indicated concentrations of EC, CH-223191, FICZ, or DHNA for 24 h were subjected to the WST8 assay (Cell Counting Kit-8, DOJINDO LABORATORIES, Kumamoto, Japan), following the manufacturer’s instructions.
For direct viable cell counting, the BMMCs (1 × 10⁶ cells/ mL) were treated with the indicated concentrations of EC, CH-223191, FICZ, or DHNA for 24 h, and the number of BMMCs was subsequently counted by a hemacytometer.
For apoptosis cells detection, the BMMCs (1 × 10⁶ cells/ mL) were treated with the indicated concentrations of EC and CH-223191 for 24 h and were evaluated by FACS using the FITC conjugated-Annexin V Apoptosis Detection Kit (DOJINDO LABORATORIES, Kumamoto, Japan), following the manufacturer’s instructions.

GBC cells were seeded at a density of 2.5×10⁴ cells/mL in 6-well plates overnight and treated with various drugs. They were then detached using trypsin-EDTA and rinsed twice with PBS. To investigate apoptosis, cells were stained with a fluorescein isothiocyanate (FITC)-conjugated annexin V apoptosis detection kit (Dojindo, Japan) according to the manufacturer's protocol and then analyzed via flow cytometry (BD, USA). For cell cycle analysis, cells were fixed with absolute alcohol at 4°C overnight. The fixed cells were centrifuged to remove ethanol, washed with PBS, and stained with DNA prep stain (Beckman Coulter, USA) at 4°C for 30 min in the dark. Finally, data were also analyzed via flow cytometry (BD, USA).