Edu dna cell proliferation kit

The proliferation of HKFs was assessed using a cell counting kit-8 (CCK-8) assay (Dojindo, Kumamoto, Japan) according to the manufacturer protocol (Wu et al., 2020 (link)). Briefly, HKFs were plated in a 96-well plate at a density of 3 × 10³/well and incubated to 40% confluence. After transfection, 10 μL CCK-8 reagent was directly added to each well of a 96-well plate at the specified time (0 h, 24 h, 48 h, 72 h, and 96 h), and then incubated for 2 h in a dark environment. Finally, a microplate reader (BioTek Instruments, United States) was applied to measure the optional density (OD) at a wavelength of 450 nm.
The proliferation of HKFs was also measured with an EdU DNA cell proliferation kit (RiboBio, Wuhan, China). After transfection, the HKFs were incubated with a medium containing 50 μM EdU for 2 h. The HKFs were fixed with 4% paraformaldehyde for 30 min, and then the excess formaldehyde was neutralized with a 2 mg/mL glycine solution. Subsequently, the HKFs were, respectively, stained in Apollo reaction cocktail and Hoechst staining solution and then incubated for 30 min in the dark. A fluorescence microscope (IX35, Olympus, Japan) was used for capturing randomly selected areas to observe the ratio of proliferating cells (EdU positive) to the total number of cells (DAPI positive) (Bieg et al., 2019 (link)). Samples were prepared in triplicate.

We assessed cell proliferation by detecting EdU (5′-ethynyl-2′-deoxyuridine) incorporation. EdU is a nucleotide analog of thymidine, and is incorporated into DNA only in proliferating cells. Following incubation with EdU for 2 hours, a fluorescent molecule from the kit that reacts specifically with EdU was added to visualize proliferating cells using a fluorescence microscope (Olympus). Using the EdU DNA Cell Proliferation Kit (Guangzhou Ribobio, Guangzhou, China) in accordance with the manufacturer's instruction, proliferating cell frequencies were calculated as the ratios of EdU-staining cells to Hoechst33342-staining cells counted from six fields of three separate experiments.

LOVO cells and HCT116 cells were divided into experimental group and control group, and were seeded in 96-well plates respectively. 1000 cells were seeded in 100 μL of medium per well and treated with 10 μL of CCK-8 solution (RiboBio, China). At 0, 24, 48, and 72 h of culture, cell absorbance at 450 nm was measured using a microplate reading element. The measurement procedure followed manufacturer's instructions (Synergy, USA).
We performed Cell-Light 5-ethynyl-2'-deoxyuridine (EdU) experiments to evaluate the proliferation capacity of cells using the EdU DNA Cell Proliferation Kit (RiboBio, China). A 24-well plate was used to seed 50,000 cells per well. One day after normal culture, cells were incubated with 50 mmol/L EdU solution for 2 h, then fixed with 4% paraformaldehyde. Following the manufacturer's protocol, we treated the cell lines with Apollo Dye Solution and DAPI, respectively, and finally captured and counted under an Olympus FSX100 microscope (Olympus, Japan).