Falcon cell strainers 40 μm

Cells (approximately 10⁶) were harvested, fixed with a 1% paraformaldehyde in phosphate-buffered saline without calcium or magnesium ions [PBS(−)]. Fixed cells were washed twice with PBS(−), and then treated for 30 min with 400 μL of 0.2 mg/mL RNase A (preheated for 10 min at 100 °C to deactivate DNase) to degrade RNA. Cells were then washed twice with PBS(−) and stained for 15 min with 0.005% propidium iodide (PI) in the presence of 0.01% NP-40 in PBS(−), which prevents cell aggregation. After filtering through Falcon^® cell strainers (40 μM) (Corning Inc., Corning, NY, USA) to remove aggregated cells, PI-stained cells were subjected to a cell sorter (SH800 Series, SONY Imaging Products and Solutions Inc., Kanagawa, Japan). Cell cycle analysis was performed with the Cell Sorter Software version 2.1.2 (SONY Imaging Products and Solution Inc., Kanagawa, Japan).

Treated and untreated Ca9-22 cells (approximately 106 cells) were harvested, fixed with 1% paraformaldehyde, treated with 0.2 mg/mL RNase A (preheated for 10 min at 100 °C to inactivate DNase), stained for 15 min with 0.01% propidium iodide in the presence of 0.01% NP-40 to prevent cell aggregation, filtered through Falcon^® cell strainers (40 μM) (Corning, NY, USA), subjected to cell sorting (SH800 Series; SONY Imaging Products and Solutions Inc., Kanagawa, Japan) and then analyzed with Cell Sorter Software version 2.1.2. (SONY Imaging Products and Solutions Inc., Kanagawa, Japan), as described previously [37 (link)].

Treated and untreated Ca9-22 cells (approximately 10⁶ cells) were harvested, fixed with 1% paraformaldehyde, treated with 0.2 mg/mL RNase A (preheated for 10 min at 100 °C to inactivate DNase), stained for 15 min with 0.01% propidium iodide in the presence of 0.01% NP-40 to prevent cell aggregation, filtered through Falcon^® cell strainers (40 μM) (Corning, NY, USA), subjected to cell sorting (SH800 Series; SONY Imaging Products and Solutions Inc., Kanagawa, Japan), and then analyzed with Cell Sorter Software version 2.1.2. (SONY Imaging Products and Solutions Inc.), as described previously [20 (link)].