Accuritm c5 cytometer

SCLC cells with or without MeCDDA treatments were collected and washed with PBS after 24-h treatment in complete medium. The cells were then fixed with 70% ice-cold ethanol at 4 °C overnight and stained with PI/RNase staining solution for 15 min in the dark for DNA profile analysis or using annexin V/PI binding kit for determining the percentages of apoptotic cells. An Accuri^TM C5 cytometer (BD Biosciences, San Jose, CA, USA) was used for both DNA profile analysis of SCLC cells and apoptosis detection.

Cells were seeded into 6-well plates (2 × 10⁵ cells/well) and treated with crassolide for 24 h. Trypsin was used to harvest the cells and then washed twice with PBS before being fixed with 70% ethanol overnight at 20 °C. The fixed cells were stained in PI solution comprising 1 mL of PBS, 50 g/mL PI (Sigma-Aldrich, St. Louis, MO, USA), 100 g/mL RNase A (Sigma-Aldrich, St. Louis, MO, USA), and 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) in the dark at room temperature. An AccuriTM C5 cytometer (BD Biosciences, Franklin Lakes, NJ, USA) was used to detect the population of cells in each phase of the cell cycle, and the results were analyzed using BD Accuri C6 Software 1.0.264.21 (BD Biosciences, Franklin Lakes, NJ, USA).

The apoptosis assay was performed using an annexin V/FITC apoptosis detection kit (BD Biosciences, Franklin Lakes, NJ, USA). The Hep3B cells (2 × 10⁵/well) were seeded in six-well plates and incubated overnight. After treatment with (−)-agelasidine A (0, 35, 70, or 140 µM) for 24 h, cells were harvested and washed in PBS. Cells were incubated with 5 μL of annexin V/FITC (20 μg/mL) and 5 μL of propidium iodide (PI) (50 μg/mL) at room temperature for 10 min in the dark. Apoptotic cells were detected using an AccuriTM C5 cytometer (BD Biosciences, Franklin Lakes, NJ, USA), and the data were analyzed using BD Accuri C6 Software version 1.0.264.21 (BD Biosciences, Ann Arbor, MI, USA).