Beyoclick edu 594 assay kit

Cell proliferation was analyzed by Cell Counting Kit-8 (CCK-8) (CellorLab, CX001L) and BeyoClick™ EdU-594 assay kit (Beyotime, C0078S). After 24h of transfection, cell suspensions were planted in 96-well plates. At 24, 48, 72 and 96h, 10ul CCK-8, and 90ul medium were incubated together at 37° C for 2h, then the absorbance at 450 nm was detected by a microplate reader.
For direct observation of the proliferating cells, the 5-Ethynyl-2-deoxyuridine incorporation experiment was also performed according to the specifications. The cells were further incubated with EdU for 2h before fixation, permeabilization, and EdU staining. The cell nuclei were stained with Hoechst33342 for 30min. Finally, the proportion of cells that incorporated EdU was detected using confocal laser microscopy.
To perform migration assays, U87 MG cells were seeded into 6-well plates. Transfection was performed when cell density reached 70-90%. 48 h later the plates were scraped with pipette tips, washed with PBS, and incubated with serum-free medium. The original images and migrated images were obtained using the inversion microscope system. The migrated area was analyzed by Image J software.

We evaluated OSCC cell proliferation using a BeyoClick™ EdU-594 assay kit (Beyotime, Shanghai, China). After cell transfection, the appropriate group was treated with inhibitors, and EdU was added to label tumor cells for 2 h. After the treatment, the cells were fixed with 4% PFA for 15 min and cleaned three times with PBS for 5 min each time. Then, the cells were treated with PBST for 15 min and washed three times with PBS for 5 min each time. The EdU reaction solution was incubated at 37 °C for 30 min according to the instructions. After washing with PBS 3 times, the nuclei were stained with a mounting medium containing DAPI for 5 min at room temperature, and cells were observed by a fluorescence microscope.

Cell proliferation ability was tested using the BeyoClick™ EDU‐594 assay kit (Beyotime) according to the manufacturer's instructions. Simply put, 10 μmol/L EDU solution was added to the culture medium, incubating for 18‐24 hours in the incubators. Labelled cells were fixed with 4% PFA at room temperature for 15 minutes. After washing three times with PBS, cells were permeabilized with 0.5% Triton X‐100 for 15 minutes. Cells were then stained with the click reaction solution containing Azide 594 for 30 minutes in the dark for fluorescence detection. Annexin V‐FITC/PI Apoptosis Detection Kit (Yeasen) was used to determine cell death. Cardiomyocytes were disassociated, resuspended and centrifuged at 300 g for 5 minutes. After that, cardiomyocytes were washed twice with pre‐cold PBS, and then, a 100 μL binding buffer was used to resuspend CMs. Next, 5 μL Annexin V‐FITC together with 10 μL PI staining solution was added and placed at room temperature for 10‐15 minutes. Finally, an extra 400 μL binding buffer was supplied and mixed thoroughly for flow cytometry.