70 μm falcon filters

The cells were seeded in 96-well plates at a density of 1 × 10³ cells per well, and a Cell Counting Kit-8 assay (CCK-8, KeyGEN BioTECH, China) was used to assess the proliferation potential. Duplicate sets of 4 wells each were assessed for each time point. At every twelve hours after seeding, the absorbance was measured at 450 nm using a plate reader (model 680; Bio-Rad, Hertfordshire, UK). The cell growth inhibition ratio (%) was calculated as follows: (1–A490 of experimental well/A490 of blank control well) × 100. Each assay was repeated at least 3 times.
The cells were collected and washed with PBS, fixed with 70% cold ethanol at 4 °C for 2 h, and passed through 70-μm Falcon Filters (BD Biosciences, Oxford, UK) to obtain a mono-dispersed cell suspension. The mono-dispersed cells were incubated with RNase A at 37 °C for 30 min and stained with propidium iodide (PI) at 4 °C for 30 min (Cell Cycle Detection Kit, BD, America). A flow-cytometric analysis was performed using a FACSCalibur flow cytometer (Becton Dickinson, Oxford, UK). Finally, the cell cycle was analyzed using Cell Quest software.

The cells were collected and washed with PBS, fixed with 70% cold ethanol at 4°C for 2 hrs, and passed through 70‐μm Falcon Filters (BD Biosciences, Oxford, UK) to obtain a monodispersed cell suspension. The monodispersed cells were incubated with RNase A at 37°C for 30 min. and stained with PI at 4°C for 30 min. (Cell Cycle Detection Kit; BD, USA). Flow cytometric analysis was performed with a FACSCalibur flow cytometer (Becton Dickinson). Finally, the cell cycle distribution was analysed by Cell Quest software (BD, Franklin Lakes, NJ, USA).