Edu apollo 488 kit

OS cell proliferation was tested by the 5-ethynyl-20-deoxyuridine (EdU) Apollo-488 Kit (Ribo-Bio, Guangzhou, China) [33 (link), 34 (link)]. OS cells with applied genetic modifications were cultured for 72h, stained with EdU (5 μM for 2h) and DAPI (for 2h). Cell nuclei were visualized under a fluorescent microscope (Leica). At least 600 cell nuclei per preparation in six random views were counted to calculate the EdU ratio (EdU/DAPI×100%).

The EdU assay was performed to assess active DNA synthesis using an EdU Apollo 488 Kit (RiboBio, China). Wild-type or stably transfected cells were seeded in 24-well coverslips at 40,000 cells per well and Sec C was added to treat the cells for 24 h. After incubation with EdU solution, the cells were fixed by 4% paraformaldehyde (Beyotime, China) and stained with Apollo 488 according to the manufacturer’s protocol (RiboBio, China). DAPI (Vector Labs, CA, USA) was used to show the nucleus. Images were acquired using a Leica SP2 confocal microscope (Leica Microsystems, Exton, PA, USA) and the ratio of EdU-positive cells was analyzed by ImageJ (Version 1.53).

The 5-ethynyl-20-deoxyuridine (EdU) Apollo-488 Kit (Ribo-Bio, Guangzhou, China) was utilized. The detailed protocols were descried early (26 (link)). Briefly, NSCLC cells were seeded into 12-well plates at 0.5 × 10⁵ cells per well and were cultured for 96h. Cell nuclei were then stained with EdU (10 μM) and DAPI, visualized under a fluorescent microscope (Leica, Beijing, China).

As previously described^{21 (link)} the 5-ethynyl-20-deoxyuridine (EdU) Apollo-488 Kit (Ribo-Bio, Guangzhou, China) was utilized for the quantification of cell proliferation. Following the applied genetic treatments, HCC cells were cultured for 48 h and stained with EdU (10 μM, 2 h at room temperature). Cell nuclei were co-stained with DAPI for 10 min, visualized under a fluorescent microscope (Leica).

EdU experiments were performed by applying an EdU Apollo 488 kit (RiboBio; C10310-1). Briefly, cells were seeded in wells of 24-well plate. The next day, cells were treated with EdU (50 μM) and kept in the incubator for 4 h. Subsequently, cells were fixed and treated with glycine, followed by permeabilization with 0.5% Triton X-100 in PBS for 5 min, and stained according to the manufacturer’s instructions.

The viability of 143B and Saos-2 cells after transfection was assessed using 5-ethynyl-2′-deoxyuridine (EdU) incorporation and colony formation assays. An EdU Apollo 488 Kit (RiboBio, China) was utilized to conduct the EdU incorporation assay. For evaluation with a Nikon inverted fluorescence microscope, EdU-positive cells were stained green, and nuclei were stained blue.
To evaluate the colony-forming ability of cells, transfected cells were counted and seeded into 6-well plates at 550 cells/well. Cells were cultured in complete DMEM supplemented with 10% FBS at 37 °C and 5% CO₂. The culture medium was replaced at 2-day intervals. After 10 days of incubation, the colonies were washed twice with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 20 min. Then, the stationary liquid was removed, and 0.1% crystal violet (Solarbio, China) was added for 20 min of staining. The 6-well plates were gently rinsed with water, and colonies with > 50 cells were counted under an optical microscope (Olympus, Japan).