Cell counting kit 8 cck 8

After 24 hours of transfection, we collected the cells and seeded them in 96-well plates at 5*10³ cells/well. All cells were incubated overnight. After 48, 72, and 96 hours of transfection, we measured the cell proliferation using Cell Counting Kit-8 (CCK-8, Roche Applied Science, USA). To determine the distribution of cell cycles by flow cytometry, we seeded the cells in six-well plates at a density of 5×10⁵/well. The experimental and control groups were assigned three wells each. Following incubation overnight, we transfected the experimental and control groups of HCC cells with sh-NC and sh-NIFK-AS1, respectively. After 48 hours of transfection, we stained the cells in the dark with a 0.025 mg/mL propidium iodide (PI) and 0.1% (v/v) Triton X-100 staining solution for DNA processing over a period of 30 minutes. Thereafter, we used an Attune™ flow cytometer (Thermo Fisher Scientific, USA) to analyze the cell cycle distribution.
To determine the apoptotic rate by flow cytometry, we collected the cells 48 hours after transfection. Next, we washed those cells with phosphate buffer saline (PBS) twice. Finally, we double-labeled these experimental cell groups with eBioscience-PI and Annexin V-FluoresceinIsothiocyanate (FITC) as described by the manufacturer. The experimental cell groups were then analyzed using the Attune™ flow cytometer.

SKOV3 and A2780 cells were plated in 96-well plates at a density of 8x10³ cells/well. After 4 h, the cells were treated with either DTX (serial dilution, 0-5 µM; Sigma-Aldrich; Merck KGaA) or TAX (serial dilution, 0-10 µM; Sigma-Aldrich; Merck KGaA) for 72 h at 37°C. A Cell Counting Kit-8 (CCK-8; Roche Diagnostics) assay was used to measure cell survival according to the manufacturer's protocol.