The cells were harvested, washed with phosphate-buffered saline (PBS) (Nacalai Tesque), and fixed in cold 70% ethanol for no less than 2 h. The cells were then washed and resuspended in 500 μL of assay buffer containing propidium iodide (PI) (Sigma-Aldrich) (20 μg/ml) and ribonuclease A (RNase A, Macherey–Nagel, Germany) (200 μg/ml) for 30 min at 20 °C in the dark. The DNA contents were analyzed using FACSVerse™ and FlowJo™ v10.8.1 (BD Biosciences, NJ, USA). A total of 10 000 events were conducted and examined as described previously59 (link).
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