Annexin 5 pe and 7 aad apoptosis detection kit

Apoptosis and necrosis was determined using Annexin V:PE and 7-AAD Apoptosis detection kit (BD Pharmingen) and flow cytometry analyses. In brief, cells were seeded and treated after 24 h with Sorafenib or DMSO. Cells were incubated for 48 h and harvested. Each cell pellet was resuspended in 100 μl of binding buffer and 5 μl of Annexin V:PE and 5 μl of 7-AAD were added. After incubation time of 15 min at room temperature, additional 100 μl of binding buffer were added. Flow cytometry analyses were performed using BD Accuri C6 flow cytometer and data thus obtained were analysed with FlowJo 10 software.

For cell cycle analysis, cells were harvested, washed twice with cold phosphate buffered saline (PBS) and fixed in 75% ethanol overnight at 4° C. The cells were then washed twice with cold PBS. Next, the cells were stained with propidium iodide solution (propidium iodide, 3.4 mM Tris-Cl, 100 μg/ml RNase A, 0.1% Triton X-100) for 20 minutes at room temperature in the dark. DNA content was then measured with a FACSCalibur flow cytometer (BD Biosciences, New York, USA), and the percentage of cells in G0/G1, S, and G2/M phase were analyzed using ModFit software (BD Biosciences). Each assay was repeated for three times.
Cell apoptosis was analyzed using Annexin V/PE and 7-AAD apoptosis detection kit (BD Biosciences). Briefly, cells were harvested after transfection, washed twice with cold PBS and resuspended in Annexin-binding buffer at a density of 10⁶ cells/ml. The cells were then stained with Annexin V/PE and 7-AAD for 15 minutes at room temperature in the dark. Cell apoptosis was detected using a FACSCalibur flow cytometer (BD Biosciences) and analyzed by Flowjo software (BD Biosciences). Each assay was repeated for three times.