Annexin 5 alexa fluor 647 pi kit

MM cells were treated with different agents and stained with Annexin V-Alexa Fluor 647/PI Kit (4A Biotech, catalog FXP023). Stained cells were examined by flow cytometry (Beckman Coulter, Navios EX).

For cell cycle detection, cells were treated with 10 μM P8 fibrils or P8 fibrils plus 50 nM 17-AAG or STA-9090 for 24 h, followed by treatment with Noc (5 nM) (MedChem Express) for another 24 h. The cells were washed with PBS and fixed with 75% ethanol at 4 °C. The fixed cells were kept at 4 °C for more than 18 h. Before staining, the cells were washed two times with PBS. Then, 200 μL PI/RNase Staining Buffers (BD Biosciences, San Jose, USA) were added to the cells and incubated for 15 min for DNA staining. Then, the cells were detected by flow cytometry. For cell apoptosis detection, cells were cultured and treated with 10 μM P8 fibrils or P8 fibrils plus 50 nM 17-AAG or STA-9090 for 24 h, followed by treatment with ActD (2 μg/mL) (MedChem Express) for another 24 h. Cells were stained with an Annexin V-Alexa Fluor 647/PI kit (#FXP023, 4 A Biotech, Suzhou, China) for apoptosis according to the manufacturer’s instructions. Apoptotic cells were quantified with a DxP Athena™ flow cytometer (Cytek Biosciences, CA, USA) and then analyzed by FlowJo V10 software. Triplicates of all assays were performed.