Beyoclick edu cell proliferation kit with tmb

The EdU incorporation assay of different cell lines was performed using a BeyoClick™ EDU Cell Proliferation Kit with TMB (Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s instructions. In brief, TE-1 and KYSE-150 cells were seeded in 96-well plates at a density of 1,000 cells per well. Cells were incubated with 10 μM EdU for 2 h at 37 °C, followed by fixation with 4% paraformaldehyde and permeabilization with 0.3% Triton X-100. Subsequently, 50 μL of Click reaction buffer containing biotin was added to each well. After incubation for 30 min at room temperature in the dark, Streptavidin-HRP working solution was added and incubated for another 30 min at room temperature. Then, cells were washed three times with PBS. After 5 min developing with the TMB developing buffer, the absorbance was measured at 630 nm using a microplate reader (Thermo Scientific, Rochester, NY, USA).

The growth of GBM cells were measured using BeyoClick™ EdU Cell Proliferation Kit with TMB (C008S, Beyotime), according to the manufacturer's instructions. Cells were then examined and counted under Fluorescence microscope.
For CCK8 assay, 1×10³ cells per well were cultured in 96-well plates and then detected using Cell Counting Kit-8 (C0037, Beyotime) reagent by measuring the absorbance at 450 nm.