Spectra max i3x multifunctional microplate detection system

Cell proliferation ability was determined using the CCK8 kit. Briefly, 1 × 10⁴ cells per well were seeded into 96‐well plates and incubated using CCK8 kit (YEASEN, Shanghai, China) at 37 °C for 4 h. The absorbance values at OD 450 nm were measured every 24 h using a Spectra Max i3x Multifunctional microplate detection system (Molecular Devices, San Jose, CA, USA). The 24-well Transwell plates (Falcon, Franklin Lakes, NJ, USA) were used for migration and invasion assay. For migration assay, 4 × 10⁴ cells per well with 200 μL Serum-free DMEM were added to the upper chamber of Transwell plates, and 500 μL DMEM medium supplemented with 20% FBS was then added to the lower chamber. After 24 h of incubation at 37 °C with 5% CO₂, cotton swabs were used to gently remove cells in the upper chamber, then fixation using 4% paraformaldehyde for 20 min and staining with 0.1% crystal violet for 10 min. Finally, images of cells were captured using microscope. For invasion assay, serum-free DMEM was first added to Matrigel (BD Biosciences, San Jose, CA, USA) in a ratio of 1:3, and then the mixture was added to upper chamber of Transwell plates and coagulated at 37 °C for 2 h. The rest of the operations were as same as the migration assay.

Cell proliferation was determined using the Cell Counting Kit-8 (TransGen Biotech Co., Ltd.), following the manufacturer's instructions. Briefly, 3,000 cells were seeded into each well of a 96-well plate. At 0, 12, 24, 48 and 72 h after seeding, each well was replaced with 100 μl fresh complete medium and 10 μl CCK-8 (TransGen Biotech Co., Ltd.) followed by incubation at 37°C with 5% CO₂ for 1 h. The absorbance of the solution was measured at 450 nm using a Spectra Max i3x Multifunctional microplate detection system (Molecular Devices, LLC). A total of three independent experiments were performed.