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The cells of interest were trypsinized and suspended with PBS, and then pelleted. After discarding the supernatant, cells were resuspended and fixed with ice-cold 70% ethanol. After 4 h, cells were pelleted and washed with ice-cold PBS, and suspended with 5 µg/ml propidium iodide (PI; Sigma-Aldrich; Merck KGaA) for 10 min in darkness at room temperature. Then, stained cells were analyzed using 3 laser Navios flow cytometers (Beckman Coulter, Inc.) and results were analyzed using FlowJo software (FlowJo LLC; version 9.7.4).
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