Cell cycle staining buffer

For cell cycle analysis, cells were harvested and fixed with −20 °C pre-cooled 70% ethanol overnight at 4 °C, followed by three washes in cold phosphate-buffered saline (PBS). Cells were stained with the cell cycle staining buffer (MultiSciences, Hangzhou, China) at room temperature for 30 minutes and analyzed with a Beckman Coulter flow cytometer (Beckman Coulter, Brea, CA, USA).

Cells were harvested in a logarithmic growth phase and seeded in six-well plates,with 10⁶ cells per well, and cultured overnight. Cell-cycle staining buffer (#CCS01; MultiSciences, Bellingham, WA, USA) was used to stain cells. Flow cytometry on a FACSCalibur system (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) was applied to analyze cell-cycle distribution.

The trypsinized A549/NCI-H1975 cells were adjusted to 3 × 10⁵ cells/mL with serum-containing DMEM/RPMI-1640 medium. The Annexin V-APC and the 7-AAD Apoptosis Detection Kit (MultiSciences, China) were used in the cell apoptosis assay. The A549 or NCI-H1975 cells were transfected with miR-1976 mimic, miR-1976 inhibitor, Lv-NCAPH lentivirus, or sh-NCAPH lentivirus for 24 h. Then, the cells were treated with serum-free DMEM/RPMI-1640 medium for 24 h to induce apoptosis. Next, the cells were stained with Annexin V-APC and 7-AAD for 30 min in the dark. Cell cycle detection was performed using cell cycle staining buffer (MultiSciences, China). Cell cycle detection was conducted when the treated cells were washed with PBS and then stained with cell cycle staining buffer for 30 min. The samples were finally tested using a flow cytometer(Becton Dickinson), and the apoptosis populations were calculated by FlowJo X software. Similarly, the cell cycle was analyzed using FlowJo X software.