Apoptosis Evaluation by Annexin-V Staining

Annexin-V staining was used to determine the proportion of apoptotic cells in accordance with a previously described experimental procedure [46 (link)]. Briefly, 1 × 10⁵ cells/well were seeded onto a 6-well cell culture plate and incubated with emodin (20 μM), sorafenib (2 μM), or combination of emodin (20 μM) and sorafenib (2 μM) for 72 h. After drug treatment, the cells were collected into fresh tubes, washed with cold PBS, and centrifuged at 2000 rpm for 2 min at room temperature. The cell pellets were incubated in 100 μL Muse™ Annexin V and Dead Cell kit reagents (Millipore, Burlington, MA, USA) for 20 min at room temperature. The Mini Flow Cytometry Muse™ Cell Analyzer (Millipore, Burlington, MA, USA) was applied for the measurement of the apoptotic cell numbers.