Quantitative Analysis of Apoptotic Cells

Apoptotic cell numbers were quantitatively analyzed using Muse™ Annexin V and Dead Cell kit (Millipore, Burlington, MA, USA) in accordance with a previously described experimental procedure [39 (link)]. Briefly, Caki-1 and A498 cells (1 × 10⁵ cells/well) were seeded and incubated for 24 h to allow stabilization, and then, 10 μM of TQ was treated for 48 h under normoxia or hypoxia. After TQ treatment, collected cells were stained with 100 μL of Muse™ Annexin V and Dead Cell kit reagents (Millipore, Burlington, MA, USA) for 20 min at room temperature. Apoptotic cell population was measured using Mini Flow Cytometry Muse™ Cell Analyzer (Millipore, Burlington, MA, USA).