Annexin 5 fitc pi

An apoptosis detection kit, annexin V‐FITC/PI obtained from eBioscience, was employed to assess cell apoptosis. Following staining protocols as per the provided instructions, the cells were examined via flow cytometry.

To assess apoptosis, OCL-AML2, OCL-AML3, MOLM-13, and MV4-11 cells were cultured and treated with different doses of ABT-199 or HHT alone or in combination for 24 h or 48 h and then double-labeled with Annexin-V-FITC/PI (eBioscience, San Diego, California, USA) for 20 min at room temperature in the dark according to the manufacturer’s instructions. The stained cells were analyzed with a NovoCyte flow cytometer (ACEA Biosciences, Inc.) with NovoExpress software. Apoptotic cells were defined as Annexin-V positive.

To assess apoptosis, CD34⁺CD38⁻ KG1α or Kasumi cells were cultured as described above for 24, 48, or 72 h with or without chidamide, Ara-C, DNR, or IDA, then double labeled with Annexin V-APC/PI (eBioscience, San Diego, CA, USA) for 15 min at room temperature in the dark according to the manufacturer’s instructions. Primary samples were stained with hCD34-APC (eBioscience, USA) and Annexin V-FITC/PI to assess the apoptosis of CD34⁺ primary cells induced by chidamide or IDA alone or the two drugs in combination. The stained cells were analyzed by flow cytometry (FACS Fortessa, BD Biosciences). Apoptotic cells were defined as Annexin V positive.

Forty-eight hours after transient transfection of KatoIII cells with miR-34a-5p mimic or negative control, the cells were collected and stained with Annexin V‑FITC/PI according to the manufacturer’s instructions (eBioscience; Cat. no. BMS500FI/100) to assay apoptosis. The apoptosis level of stably transfected cells was assessed using Annexin V‑PE/7-AAD (BioLegend; Cat. no. 640,908) in addition to Annexin V‑FITC/PI staining. Flow cytometry was performed to detect the apoptosis level of the transfected cells. The data was analyzed by FlowJo software 7.6.1 (Tree star, Inc., San Carlos, CA, USA).

Flow cytometric analysis of HL-60 cells double-stained with apoptosis annexin V-FITC/PI (eBioscience, Vienna, Austria) was used to determine cell apoptosis. Briefly, HL-60 cells were seeded in 24-well plates and incubated with e-As₄S₄/1:15–60 at different concentrations of As₄S₄ for 6–48 h. After centrifugation and washing with PBS, cells were resuspended in binding buffer. Then, annexin V-FITC and PI were added, followed by a 10-min incubation at room temperature, and were analyzed using a flow cytometer (Accuri C6; BD Company, Franklin Lakes, NJ). In total, 1.5 × 10⁴ cells were measured and the data acquired were analyzed with the CFlow Plus software.
Apoptotic cells were also examined by microscopy following Hoechst 33342 (Beyotime Biotechnology, Haimen, China) staining. Cells were seeded in 24-well plates and a suspension of e-As₄S₄/1:15–60 at 40 mg/L of As₄S₄ was added. After 48 h incubation, the cells were harvested in a 1.5-mL concentration tube, washed twice with PBS, and suspended in 1 mL medium. Hoechst 33342 at 5 μL/mL was added to the tube and incubated for 25 min at 4 °C in the dark. Then the cells were washed twice with PBS to remove unbound dye and imaged with a fluorescence microscope (IX71, Olympus, Tokyo, Japan).