5 ethynyl 2 deoxyuridine edu labeling detection kit

Cell vitality was detected using the Cell Counting Kit-8 (CCK-8) assay kit (Bimake). Briefly, transfected cells were seeded into 96-well plates and incubated with CCK-8 reagent every 24 hours according to the manufacturer’s instructions. For colony formation assay, cells after transfection were cultured in 6-well plates, and after 14 days, the cells were fixed with methanol and stained with 0.1% crystal violet. Visible colonies were photographed. Cell proliferation was identified using Ethynyldeoxyuridine (EdU) assay following the manufacturer’s protocol of 5-ethynyl-2-deoxyuridine (EdU) labeling/detection kit (Ribobio, Guangzhou, China). The transfected cells were treated with 50 µM Edu labeling medium and incubated for 2 hours. Next, the cells were fixed with 4% paraformaldehyde and the cell membrane was permeated with 0.5% Triton X-100. Subsequently, cells were added with anti-Edu working solution and DAPI staining solution. Edu positive cells were identified and counted under fluorescent microscopy. The percentage of Edu positive cells was calculated from five random fields in three wells.

Proliferating cells were assessed using a 5-ethynyl-2-deoxyuridine (EdU) Labeling/Detection Kit (RiboBio, Guangzhou, China) according to the manufacturer’s protocol. The GC cells were cultured in 96-well plates at a density of 1x10⁴ cells per well and transfected with the circGRAMD1B overexpressing plasmid or the siRNA1/siRNA2 for 48 h. Then, 50 μl/L EdU labeling media were added to the plates, which were incubated for 2 h at 37 °C under 5% CO₂. After treatment with 4% paraformaldehyde and 0.5% Triton X-100, cells were stained with the anti-EdU working solution. Nuclei were stained with Hoechst 33342 reaction solution. The percentage of EdU-positive cells was calculated from five random fields in three wells under a confocal laser scanning microscopy (LSM800, Carl Zeiss AG, Germany).

Based on the protocol outlined in the manual of the 5-ethynyl-2-deoxyuridine (EdU) labeling/detection kit (RiboBio, Guangzhou, PR, China), Cells of the control and experimental groups were digested and inoculated into a 96-well plate. After 24h of culture, labeling medium with 50 μM of EdU was added to the cell culture, and was incubated for 2 h at 37 °C with 5% CO₂. The cells were then fixed with 4% paraformaldehyde (pH 7.4) for 30 min and incubated with glycine for 5 min. After being washed with PBS, cells were stained with anti-EdU working solution at room temperature for 30 min. They were then washed with 0.5% Triton X-100 in PBS, and incubated with DAPI at room temperature for 3 min. Cells were then observed using fluorescent microscopy.