Cells were fixed with 70% ethanol and incubated at 4 °C overnight. RNase A stock solution (final concentration: 0.5 mg/mL) was added and incubated for 20 min at 37 °C. Propidium iodide (PI, 50 mg/mL; Sigma-Aldrich; P4170) was added and incubated for 15 min at 4 °C in the dark. Cell cycle was measured by fluorescence-activated cell sorting (FACS) using a BD FACSCalibur™ HG Flow Cytometer Instrument (BD, Franklin Lakes, NJ, USA) and Cell Quest Pro software v3.1 (BD). FlowJo® v10 (BD Biosciences, San Jose, CA, USA) was used for the analysis. The experiment was repeated three times [24 (link)].
Kukita A., Sone K., Kaneko S., Kawakami E., Oki S., Kojima M., Wada M., Toyohara Y., Takahashi Y., Inoue F., Tanimoto S., Taguchi A., Fukuda T., Miyamoto Y., Tanikawa M., Mori-Uchino M., Tsuruga T., Iriyama T., Matsumoto Y., Nagasaka K., Wada-Hiraike O., Oda K., Hamamoto R, & Osuga Y. (2022). The Histone Methyltransferase SETD8 Regulates the Expression of Tumor Suppressor Genes via H4K20 Methylation and the p53 Signaling Pathway in Endometrial Cancer Cells. Cancers, 14(21), 5367.
Incubation of cells with 70% ethanol at 4 °C overnight
Addition of RNase A stock solution (final concentration: 0.5 mg/mL) and incubation for 20 min at 37 °C
Addition of propidium iodide (PI, 50 mg/mL) and incubation for 15 min at 4 °C in the dark
dependent variables
Cell cycle measured by fluorescence-activated cell sorting (FACS) using a BD FACSCalibur™ HG Flow Cytometer Instrument and Cell Quest Pro software v3.1
control variables
Cell fixation with 70% ethanol
Incubation temperature and duration for RNase A and propidium iodide treatments
Annotations
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