5 ethynyl 2 deoxyuridine edu assay

Cell proliferation in situ was analyzed with 5-ethynyl-2′-deoxyuridine (EdU) assays from Invitrogen. EdU is a thymidine analogue and incorporated into the DNA of dividing cells. Mice were injected intraperitoneally with 100 μg of EdU in PBS on day 6 after wounds and then the skin specimens were collected one day later20 (link). Two specimens were excised from each wound, one from the center of the wound bed and the other from uninjured adjacent skin served as reference such that a total of 4 specimens were collected from each mouse. The skin specimens were formalin-fixed, embedded in paraffin, and cut into 5μm thick sections. For EdU staining, paraffin in the tissue sections was removed and the tissue was stained with the Click-iT^® EdU Alexa Fluor^® 488 Imaging Kit (Invitrogen, USA), counterstained with DNA dye DAPI (4′,6-diamidino-2-phenylindole) provided in the kit per the manufacturer’s instruction. The stained sections were imaged by confocal microscopy (Olympus FV1000, Olympus, Japan) and analyzed by Image J software. Percentages of EdU-positive cells relative to a total number of cells were obtained by manually counting the number of EdU-positive green cells and DAPI-positive blue cells in 100 microscopic fields randomly selected in each sample in a sample-blind manner.

After 48 h transfection, CAL27 and SCC25 cells (2 × 10⁴/well) were seeded in 24-well plates. The 5-ethynyl-2′-deoxyuridine (EdU) assay (Life Technologies Corporation, USA) was used to evaluate the proliferation ability of OSCC cells as previously reported [16 (link)]. Briefly, CAL27 and SCC25 cells were incubated with 100 μL EdU reagent for 2 h at 37 °C, and stained with DAPI and visualized by a fluorescence microscope (Olympus, Tokyo, Japan).