Bovine pancreatic ribonuclease

Cells (0.5 × 10⁴) were harvested by trypsinization, washed in ice-cold phosphate-buffered saline, and fixed in 80% ice-cold ethanol in phosphate-buffered saline. Before staining, cells were gently precipitated and resuspended in cold phosphate-buffered saline. Bovine pancreatic ribonuclease (Sigma-Aldrich, St. Louis, MO, USA) was added to a final concentration of 2 μg/ml, and cells were incubated at 37°C for 30 min, followed by incubation with 20 μg/ml propidium iodide (Sigma-Aldrich) for 20 min at room temperature. Cell samples (5 × 10⁵) were then analyzed by a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA).

CD34+CD38− LSCs were harvested and washed twice in PBS. CD34+CD38− LSCs were fixed overnight using 70% ice-cold ethanol (Beyotime Biotech., Shanghai, China), and washed twice using PBS. Then, the CD34+CD38− LSCs were treated using bovine pancreatic ribonuclease (Sigma-Aldrich, St. Louis, MO, USA) at a dose of 1 mg/ml for 30 min at 37°C. The cells were stained with the propidium iodide (PI) at a concentration of 50 μg/ml in the dark for 30 min. The stained CD34+CD38− LSCs were viewed and imaged using the FACS Aria IIU flow cytometer (BD Bioscience, Oxford, UK) and the percentages of cells in each phase of the cell cycle were analyzed using MultiCycle software (Phoenix Flow Systems, Tokyo, Japan).