Cell light edu dna cell kit

A cell proliferation assay was performed using an MTT and 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay. For the MTT assay, cells at a density of 5x10³ cells/well were incubated in 96-well plates in triplicate and cultured at 37˚C for five days. At the timepoints of 1, 2, 3, 4 and 5 days, cells were treated with 10 µl MTT (0.5 mg/ml; Sigma-Aldrich; Merck KGaA) for 2 h at 37˚C, followed by incubation with 100 µl of DMSO for 2 h at 37˚C. The absorbance was recorded at 595 nm using an enzyme immunoassay analyzer (Bio-Rad Laboratories, Inc.). For the EdU labeling assay, a Cell-Light EdU DNA cell kit (Guangzhou RiboBio Co., Ltd.) was used. Transfected cells were seeded into 96-well plates (1x10⁴ cells/well) and cultured at 37˚C for 48 h. Subsequently, cells were washed twice with PBS and permeabilized using 0.5% Triton X-100 for 10 min. Subsequently 50 mM EdU solution was added to each well for 2 h, and the cells were fixed and stained using 1x Apollo567 for 30 min in darkness at 37˚C. The nucleus was counterstained with DAPI (Sigma-Aldrich; Merck KGaA) at 37˚C for 30 min. Stained cells were examined using a fluorescence microscope (magnification, x200; Nikon Corporation). The proportion of EdU-positive nuclei (red) to blue fluorescent nuclei was calculated by counting six microscopic fields randomly for each well in five separate experiments.

SGC‐7901 cells were seeded on 24‐well plates and transfected with miR‐155 mimics, inhibitors, TGFβR2 siRNA, and the relevant negative controls. A Cell‐light EdU DNA cell kit (Ribobio, Guangzhou, China) was used to measure the cell proliferation ability at 24 hour post‐transfection, following the manufacturer's instructions. Cells were incubated with 50 μmol/L EdU for 5 hours and fixed within 4% paraformaldehyde for 30 minutes at room temperature. The cells were washed with PBS twice and treated with 0.5% Triton X‐100 for 10 minutes. Subsequently, the cells were incubated in the dark with Apollo (Ribobio) for 30 minutes and in Hoechst 33342 (Ribobio) for a further 30 minutes. All of the staining was carried out in triplicate.

After the cells were seeded in 24‐well plates, 5‐ethynyl‐2′‐deoxyuridine (EdU) was added to the culture medium at a concentration of 50 μM/mL for 8 h to chase the DNA template according to the instructions in the Cell Light EdU DNA Cell Kit (Apollo 488/567, RiboBio). Briefly, after being fixed in 4% paraformaldehyde and treated with 0.5% Triton X for 15 min, the cells were incubated with Apollo in the dark, and the nuclei were stained with Hoechst 33342. EdU‐labeled cells were counted manually in five fields of view randomly selected from each well, and the percentage of positively stained cells was calculated.

The effects of honokiol on TGF-β1-induced proliferation were determined using a Cell-Light EdU DNA cell kit (Guangzhou RiboBio Co., Ltd.,), according to the manufacturer's instructions. In brief, after being subjected to the appropriate treatments, fibroblasts were incubated at 37°C in EdU solution (50 µmol/l) for 2 h. The cells were then fixed in 4% formaldehyde for 30 min and incubated at room temperature with 0.5% Triton X-100 in PBS for 10 min. After washing with PBS, Click-iT™ reaction cocktail was added to the plates for 30 min, and the cells were then stained with Hoechst 33342 dye at room temperature for a further 30 min. Proliferating cells were imaged under a fluorescent microscope (Nikon Corporation) and were counted using ImageJ software (NIH). A total of 5 fields were randomly selected for microscopic observation.