Muse annexin 5 and dead cell kit reagents

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).

The apoptotic cell population was measured by using Muse™ Annexin V and Dead Cell kit (Millipore, Burlington, MA, USA) in accordance with a previously described [51 (link)]. Initially, LEF1 stably overexpressing A375 and A2058 cells were generated with puromycin selection. Empty vector (pBABE-puro-empty vector) or LEF1 (pBABE-puro-LEF1) overexpressing A375 and A2058 cells (1 × 10⁵ cells/well) were seeded onto a 6-well cell culture plate and incubated for 48 h in the absence or presence of 100 nM of cinobufagin. After cinobufagin treatment, the cells were harvested and washed with cold PBS, and then cells were incubated in 100 μL Muse™ Annexin V and Dead Cell kit reagents (Millipore, Burlington, MA, USA) for 20 min at room temperature. Finally, the cells were subjected to Mini Flow Cytometry Muse™ Cell Analyzer (Millipore, Burlington, MA, USA) to measure apoptotic cell populations.

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.