5 ethynyl 2 deoxyuridine edu assay kit

Tumor cell proliferative capacity was evaluated using the 5-ethynyl-2′-deoxyuridine (EdU) assay kit (RiboBio). Twenty-four hours after transfection, the cells were inoculated into 96-well plates at a density of 8 × 10³ cells per well, and after subsequent 24 h incubation, the cell samples were incubated with 50 µM EdU at 37°C for 2 h and subsequently fixed with 4% paraformaldehyde for 30 min. The fixed cells were rinsed with 2 mg/mL glycine for 5 min, and phosphate-buffered saline (PBS) with 0.5% Triton X-100 was subsequently applied for 10 min. The cells were stained with 1× Apollo staining solution for 30 min. After rinsing with PBS (0.5% Triton X-100) for 10 min, the cells were incubated in 100 mL of 1× Hoechst 33,342 for 30 min. Subsequently, the cells were fixed in 4% paraformaldehyde and stained with DAPI. Finally, cell counting was performed using fluorescence microscopy to quantify the percentage of EdU-positive cells [32 (link)].

The 5‐Ethynyl‐2’‐deoxyuridine (EdU) assay kit (RiboBio, Guangzhou, China) was employed to evaluate cell growth ability. The transfected cells, cultured in a well of a 96‐well plate at a density of 5000 cells per well, were labeled with 50 μm of EdU and incubated for additional 2 h before being fixed with 4% paraformaldehyde (pH = 7.4) for 30 min. Then, 0.5% Triton X‐100 was added to each well and incubated at room temperature for 10 min. After that, anti‐EdU working solution (Apollo^R reaction cocktail) and Hoechst 33342 were successively employed to stain the cells. The EdU‐labeled root cells were observed under a fluorescence microscope (Nikon, Tokyo, Japan).

Approximately 1.0 × 10⁴ transfected SCC-4 and CAL-27 cells were cultured in 96-well plates, and then underwent 1-h incubation with CCK-8 reagent (Beyotime, Shanghai, China). The absorbance at 450 nm was recorded using an Infinite M200 multimode microplate reader (Tecan, Shanghai, China).
After approximately 48-h transfection, the 5-ethynyl-2’-deoxyuridine (EdU) assay kit provided by Ribo (Guangzhou, China) was utilized to examine the proliferation of SCC-4 and CAL-27 cells. Specifically, cells were grown in culture medium containing EdU (Invitrogen) solution (1:1,000). At the proliferative stage, the cells were labeled with EdU for 2 h, followed by rinsing with PBS (0.5 g/ml) thrice. Subsequently, the cells were stained by 4′,6-diamidino-2-phenylindole (DAPI) from Invitrogen for 10 min at indoor temperature in the dark and underwent PBS rinsing more than twice. Ultimately, assessment of the stained cells was implemented via the FACSCalibur DxP flow cytometer (BD Biosciences, Shanghai, China).

For viability assays, cells were plated at 1 × 10⁴ cells/well in triplicate. Assays were performed using CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega) or Cell Counting Kit-8 (CCK-8) (Dojindo). Cell proliferation was also measured using the 5-ethynyl-2'-deoxyuridine (EdU) assay kit (RiboBio) according to the manufacturer's instructions. For the cell cycle analysis, the cells were fixed and then stained with PI staining buffer (Multisciences Biotech) for 30 min at room temperature in the dark, and measured using flow cytometry. Soft agar colony formation assays were performed as previously described. Briefly, the 5 × 10⁴ cells in 0.4% Noble agar were plated on top of the 0.8% Noble agar bottom layer in a 6-well plate. After 3 weeks, they were stained with 1 mg/ml p-iodonitrotetrazolium chloride for visualization and counting. In addition, mouse serum was collected and measured using an ELISA kit (Immunodiagnostic Systems) according to the manufacturer's instructions.

The 5-ethynyl-2′-deoxyuridine (EDU) assay kit (Guangzhou RiboBio, Guangzhou, China) was used to monitor cell proliferation. The cells were inoculated into 96-well plates (1 × 10⁴/well) until 80% confluence. Each well was added with 100 μl of EDU solution and incubated for 2 h. The cells were incubated with 4% paraformaldehyde at room temperature for 30 min. Then, the cells were treated with 100 μl of 1× Apollo^® staining reaction solution for 30 min. Next, 100 μl of Hoechst 33342 reaction solution was added to each well and incubated for 30 min. A microscope (DSZ2000X, Beijing Cnmicro Instrument Co., Ltd., Beijing, China) was used to observe and take pictures.