Annexin 5 fitc

After transfection, 4 × 10⁶ cells were treated with trypsin. After washing with precooled PBS buffer without calcium and magnesium, cells were mixed with 100 μl binding buffer which followed by incubation for 10 min in the dark. After that, 6 μl of Annexin V-FITC and 10 μl of PI stain (MA0220, Meilun Bio, China) was added and cells were incubated in dark for 20 min. Finally, apoptotic cells were detected by flow cytometry.

The breast cancer cells MDA-MB-231 were collected and subjected to the following experiments in strict accordance with the instructions of Annexin V-FITC/propidium iodide (PI) apoptosis detection kit (MA0220, Meilunbio, Dalian, China). Initially, the cells were counted. Next, 2–5 × 10⁵ cells/mL were centrifuged at 500 × g for 5 min. After the supernatant had been discarded, the cells were resuspended with 195 μL binding buffer working solution, incubated with 5 μL Annexin V-FITC for 10 min, and later 10 μL PI (20 μg/mL) for 5 min under conditions devoid of light. The blank control, PI simple staining and Annexin V-FITC single staining groups were established.

Myoblasts were washed twice with PBS, and the concentration was adjusted to 10⁶ cells/mL. The 100 µL of binding buffer (MeilunBio, Dalian, China) was then added to the cell suspension along with 5 µL of annexin V–FITC (MeilunBio), cells were then incubated for 10 min at room temperature. Cells were then incubated with 7.5 µL of propidium iodide (PI, MeilunBio) for 15 min at room temperature in the dark. Apoptosis was analyzed by fluorescence microscopy, as well as by flow cytometry using FlowJo software (v7.6.1, Ashland, OR, USA).