Evaluating Cell Proliferation Assays

A CCK-8 kit was used to assess the proliferation of cells at specific time points based on provided instructions. Briefly, cells were seeded in 96-well plates (2 × 10³/well), followed by the addition of CCK-8 solution to each well and incubation for 15 h. Absorbance at 450 nm was then assessed with a spectrophotometer. Colony formation assays were conducted by plating cells in 6-well plates (800/well) and incubating them for 14 days, followed by fixation with 4% paraformaldehyde (PFA) and staining with 0.5% crystal violet. EdU uptake was assessed as reported previously [11 (link)]. Briefly, cells were added to 96-well plates (1 × 10³/well) for 24 h, after which EdU (50 μM) was added, and cells were fixed for 15 min with 4% formaldehyde prior to permeabilization for 20 min using 0.5% Triton X-100. After rinsing with PBS, 100 μL of 1× Apollo reaction cocktail was added, cells were incubated for an additional 30 min, and they were then imaged via fluorescent microscopy (Leica, Germany).