Before cell cycle analysis, cells were subjected to cell cycle synchronization. Synchronization was performed as described previously.
17 (link) Cells were detached using 1 mL of trypsin (0.25%), collected in Eppendorf (EP) tubes, and centrifuged for 5 min (1500 rpm), and the supernatant was then discarded. The cells were fixed with 500 μL of 75% ethanol for 2 h and were then centrifuged, washed twice with PBS, and centrifuged again. The supernatant was then discarded. The cells were incubated with 500 μL of propidium iodide (PI)/RNaseA working solution in the dark at room temperature for 60 min. Fluorescence signals were detected and recorded at an excitation wavelength of 488 nm with a FACS system (Becton Dickinson) running Cell Quest research software (Becton Dickinson).
17 (link) Cells were detached using 1 mL of trypsin (0.25%), collected in Eppendorf (EP) tubes, and centrifuged for 5 min (1500 rpm), and the supernatant was then discarded. The cells were fixed with 500 μL of 75% ethanol for 2 h and were then centrifuged, washed twice with PBS, and centrifuged again. The supernatant was then discarded. The cells were incubated with 500 μL of propidium iodide (PI)/RNaseA working solution in the dark at room temperature for 60 min. Fluorescence signals were detected and recorded at an excitation wavelength of 488 nm with a FACS system (Becton Dickinson) running Cell Quest research software (Becton Dickinson).
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