Click it edu cell proliferation imaging kit

5000 cells/well were seeded in a 96 well plate for 12–16 hr after which the cells were incubated with 10 μM EdU (5-ethynyl-2’-deoxyuridine) for 6 hr. EdU incorporation was performed using the instructions provided in the Click-iT EdU Cell Proliferation Imaging Kit (Invitrogen, C10337). EdU incorporation was also analyzed using Click-iT EdU Alexa Fluor 488 Flow Cytometry Assay Kit (Invitrogen, C10420) after 24 hr incubation with 10 μM EdU. The cells were then trypsinized, permeabilized and stained as per kit instructions. Cell viability assay was performed using the Apoptosis Assay Kit (Cat# 22837).

OCSCC cell proliferation was analyzed using the Click-iT™ EdU Cell Proliferation Imaging Kit (Alexa Fluor™ 647) (C10340, Thermo Fisher). The cells were seeded into 6-well plates and incubated for 12 h before adding 10 µM EdU for 1 h at 37 °C in 5% CO₂. After fixation using 4% paraformaldehyde, the cells were permeabilized with PBS containing 0.5% Triton X-100 at room temperature for 20 min and incubated with Click-iT ® reaction mixture with fluorescent labeling for 20 min. After labeling the nuclei with DAPI, the number of proliferating cells was observed under a fluorescence microscope, and the positive rate of EdU (%) was counted.
The cells were seeded into 6-well plates at 500 per well and incubated at 37 °C with 5% CO₂ for two weeks, followed by fixation of the cells with 4% paraformaldehyde for 15 min and staining with crystal violet for 10 min. The number of cell colonies formed was counted under a light microscope.