Beyoclick edu 488 cell proliferation kit

EdU assay was implemented for detecting the proliferation of Huh7 and HCCLM3 cells according to the instructions of the BeyoClick™ EdU-488 Cell Proliferation Kit (Beyotime, Shanghai, China). The cells were inoculated on 24-well plates with 2 × 10⁵ cells per well. 200 μL of 5 μmol/L EdU working solution was added to each well and incubated for 2 h, and the cells were rinsed with PBS. The cells were fixed with 4% paraformaldehyde and then incubated with 200 μL of glycine (2 mg/mL) for 5 min and cleared with PBS on a shaker for 5 min. Afterward, 100 μL 0.5% Triton X-100 was added for permeability. Subsequently, the cells were incubated with the click additive solution (in the dark, RT, 30 min) and then stained with 1 × Hoechst 33342 DNA staining solution (in the dark, RT, 20 min). After washing with PBS, the cells were photographed and calculated under a fluorescence microscope (Olympus, Tokyo, Japan).

Cell viability and proliferation was measured by using Cell Counting Kit-8 (CCK-8) assay and EdU staining assay, respectively [35 (link)]. For CCK-8 assay, cells with different treatments were seeded into the 96-well plates at a density of approximately 4 × 10⁴ cells/well. Next, 10 µL of CCK-8 solution (MedChemExpress, USA) was added into each well and then were incubated at 37 °C for 1 h, then the absorbance of each sample was determined at 450 nm by using a microplate reader (Thermo Fisher Scientific). EdU staining assay was performed using BeyoClick™ EdU-488 Cell proliferation kit (Beyotime, China) according to the manufacturer’s instructions. Briefly, cells with different treatments were incubated with Edu working solution for 2 h and then fixed with 4 % paraformaldehyde (PFA) for 15 min at room temperature. After permeabilized with 0.3 % TritonX-100 for 15mim at room temperature, the cells cultured with 500 µL Click Additive Solution for 30mim at darkness. Subsequently, cell nucleus was counterstained with DAPI for 10mim. Finally, the EdU-positive cells were then observed under the fluorescence microscope, and the percentage of EdU-positive cells was calculated as the cell proliferation rate.

The EdU incorporation assay was performed using the BeyoClick EdU-488 Cell Proliferation Kit (Beyotime, Shanghai, China). EdU (10 μM) was added to the cell medium and cultured for 2.5 h. After fixation with 4% paraformaldehyde and permeation with 0.3% Triton X-100, the cells were incubated with a click reaction mixture at room temperature for 30 min in dark. Subsequently, cell proliferation was detected by flow cytometry (BD Biosciences, FACSCanto II, USA).

U-118 MG cells were stained using BeyoClick™ EdU-488 Cell Proliferation Kit (Beyotime, Shanghai, China). In brief, U-118 MG cells (1.5 × 10⁵ cells/well) were seeded in a 6-well plate, transfected with NC or si-MSH2, and cultured in an incubator at 37°C. After 96 h of transfection, U-118 MG cells were incubated with EdU for 3 h, fixed with 1 ml paraformaldehyde (4%) for 15 min, and permeabilized with 0.3% Triton X-100 (Beyotime) for 15 min. Next, the U-118 MG cells were incubated with 500-µL of the click reaction mixture for 30 min in the dark, washed three times, and incubated with Hoechst buffer for 15 min.

Primary chondrocytes were seeded in 96-well plates, pre-treated with 10 ng/ml IL-1β for 24 h, and then incubated with MON (0, 25, 50 and 100 μM) for 24 h. Cell proliferation was assayed using BeyoClick™ EdU-488 Cell Proliferation Kit (Beyotime, Beijing, China) [38 (link), 40 (link)]. The EdU-labeled cells were observed under a fluorescence microscope (Olympus IX73), and the proportion of proliferating cells were calculated using Image J.

The LDLR−/− mice were fed a high-fat diet (HFD) for 8 weeks, with or without evodiamine treatment, and EdU (50 mg/kg, Beyotime, Shanghai, China) was intraperitoneally injected for 4 h. After being anesthetized with 5% isoflurane for 5 min, and 1 min after breathing stopped, the mice were euthanized by decapitation. The aortic root tissue was fixed in 4% PFA and embedded in paraffin. Paraffin sections (5 μm) were dewaxed and dehydrated. Antigen retrieval was performed using citrate buffer; samples were then permeabilized and blocked in 3% bovine serum albumin (BSA) at room temperature for 1 h. The sections were incubated with primary antibodies in the blocking buffer overnight at 4 °C, and immunofluorescence staining was performed with the corresponding secondary antibody (1:500) (Thermo Fisher Scientific, Waltham, MA, USA). The EdU reaction solution was prepared accordingly using the manufacturer’s instructions (BeyoClick™ EdU-488 Cell Proliferation kit, Beyotime, Shanghai, China). The tissues were then incubated with the EdU reaction solution for 30 min at room temperature and protected from light.
The slides were stained with Hoechst 33,342 and washed with 3% BSA-PBS. Images were acquired with an AxioScopeA1 light microscope (Carl Zeiss, Jena, Germany).