Cck 8 reagent

The Cell Counting Kit (CCK)-8 assay was used to detect whether cells were proliferating. Trypsin was used to initially break down the cells (Servicebio Technology, Wuhan, China), after which they were resuspended in media. Then, 1000 cells were plated onto 96-well plates. Phosphate-buffered saline (PBS) was used to wash the cells after the growth media was removed. On days 1, 2, 3, 4, and 5, we applied 10 μL of the CCK-8 reagent (Servicebio Technology) to each well. Cell viability was assessed using a microplate reader (Bio-Rad Laboratories Inc., Hercules, CA, USA) to measure absorbance at 450 nm after 1 h of incubation in complete darkness.

Osteosarcoma cells were trypsinized and resuspended in a complete medium. For the CCK-8 assay, cells were cultured in 96-well plates at an initial density of 3 × 10³ cells/well. CCK-8 reagent (Servicebio Technology) was added into each well at 0, 24, 48, and 72 h. After incubation for 1 h at 37 °C, the absorbance readings were obtained at 450 nm. For the colony formation assay, cells were seeded into six-well plates at an initial density of 5 × 10² cells/well and were cultured for ~10 days. Colonies were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. For the transwell invasion assay, 1 × 10⁵ cells in 200 μl serum-free medium were added into the upper well of a transwell chamber (Corning Costar, Corning, NY, USA) precoated with Matrigel, and 600 μl medium containing 20% FBS was added into the lower chamber. Invaded cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet and observed by inverted microscopy (Olympus, Tokyo, Japan).

The Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2 (EDU) assays were performed to measure cell proliferation. For the CCK-8 assay, PC cells were seeded at a density of 3000 cells/well in a 96-well plate. Following cell adhesion for 0, 24, 48, 72, and 96 h, 100 μL of fresh DMEM containing 1/10 CCK-8 reagent (Servicebio, Wuhan, China) was added to each well. After culturing for 2 h, the absorbance (OD) of each well was measured at 450 nm. The EDU assay was performed using the BeyoClick™ EdU-488 Cell Proliferation Kit (Beyotime, Shanghai, China) according to the manufacturer’s protocol. The EDU-positive cell index was determined by calculating the ratio of EDU-positive cells to total cells using ImageJ software (version 1.8.0, MD, USA).