Apoptosis and Cell Cycle Analysis of ZT55 in HEL Cells

Apoptosis of ZT55-treated cells was assessed by FACS analysis after staining with Annexin-V and propidium iodide (PI) [18 (link)]. HEL cells (10⁶ cells/ml) were treated for 24, 48 or 72 h with different concentrations of ZT55 (0, 12.5, 25, or 50 μM). The cells were then harvested, washed twice with ice-cold PBS, and stained with Annexin-V FITC (BD Biosciences, Franklin Lakes, NJ, USA) for 15 min at room temperature. Cells were then washed with PBS to remove unbound antibody and PI (BD Biosciences) was added. Samples were analyzed with a FACS BD verse cytometer (BD Biosciences).
For cell cycle assay, HEL cells were seeded at a density of 5 × 10 ⁵ cells/ml and treated for 24 h with the serial concentration of ZT55. After treatment, cells were fixed with 70% ice-cold ethanol, stained with 20 ng/ml of PI and analyzed with a FACS BD verse cytometer [19 (link)].
To characterize expanded MPN blasts, cells were labeled with monoclonal antibodies against CD34⁺ (BD Biosciences) and analyzed with a FACS BD verse equipped with the CellQuest Pro software (BD Systems, San Jose, CA, USA) [20 (link)].