Beyoclick edu cell proliferation kit with alexa fluor 555

Resuspended transfected cells were seeded in 96-well plates, and cultured at a density of 2 × 10⁴ cells per well for 24 h. Cell EdU labeling, immobilization, purifying, EdU detecting and nuclear staining were carried out according to the instructions of the BeyoClickTM EdU Cell Proliferation Kit with Alexa Fluor 555 (Beyotime, China).
Cell proliferation was observed and analyzed by a high throughput microplate imager (Operetta CLS®™, PerkinElmer, Beijing, China).

HepG2 cells were fixed in 4% paraformaldehyde at room temperature for 15 min, and these HepG2 cells were blocked with 5% BSA containing 0.3% TritonX-100 buffer at room temperature for 60 min. The primary antibodies used are listed in Table S2. Secondary antibodies containing either FITC or Cy3 (Beyotime, Shanghai, China) were used using immunofluorescence staining. An inverted microscope was used to analyze cell staining. For cell proliferation analysis, HepG2 cells were processed using the BeyoClickTM EdU Cell Proliferation Kit with Alexa Fluor555 (Beyotime, Shanghai, China) kit according to the instructions.

For the colony-formation assay, 1 × 10³ lung-cancer cells were seeded into six-well plates, and 24 h later the cells were treated with JAC4 at the indicated doses and cultured for 10–14 days. The medium was changed every 3 days, then fixed with 4% paraformaldehyde and stained with crystal violet (Beyotime, Shanghai, China). Visualize colonies were counted. For EdU-staining assays, proliferating cells were detected with the BeyoClick^TM EDU Cell Proliferation Kit with Alexa Fluor 555 (Beyotime, Shanghai, China) according to the manufacturer’s protocol. Cells were seeded in 96-well plates and incubated for 2 h with EDU working solution (20 μM) after the cells had returned to the normal state, followed by fixation with 4% paraformaldehyde for 30 min. Then, they were incubated with PBS containing 0.3% Triton X-100 for 15 min at room temperature. To detect the percentage of cell proliferation, the cell nuclei were stained for 10 min using 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI). Next, images of cells were acquired with a Nikon Ti microscope (Nikon, Tokyo, Japan).