Beyoclick edu cell proliferation kit with alexa fluor 594

Cells transfected as before were cultured in 96-well plates with 6000 cells in each well. The cell viability was assessed using the Cell Counting Kit-8 (Cat# 40203ES80, Yeasen) at 24, 48, and 72 h, respectively. All CCK8 assays were performed in triplicates.
The proliferation of cells was tested using a BeyoClick^™ EdU Cell Proliferation Kit with Alexa Fluor 594 (Cat# C0078S, Beyotime Institute of Biotechnology, Shanghai, China). The cells were exposed to 50 μM Edu for 2 h at 37 °C following the manufacturer’s instructions. The EdU-stained and DAPI-stained cells were visualized by fluorescence microscopy (Olympus). The analysis of cell proliferation was performed using images of randomly selected fields obtained from the fluorescence microscope. We performed three repeats for each group, and three images were used to calculate the cell proliferation rate in each repeat.

Cell proliferation was determined by the CCK-8 Kit (Dojindo Laboratories) according to the manufacturer’s instructions. Briefly, 10 μL of CCK-8 solution was added to cultured cells in each well, followed by incubation at 37°C for 1 h. The OD values were measured at 450 nm using a microplate reader. EdU staining was conducted using the BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 594 (Beyotime, Cat. No: C00788L). Cells were washed with PBS. Fresh DMEM was added, and then, 10 µM EdU was added into the medium. The cells were incubated for 2 h at 37°C/5% CO2. After the incubation, the cells were washed with PBS to remove the DMEM and the free EdU probe. The cells were then fixed in 4% paraformaldehyde at room temperature for 30 min before being stained with DAPI for 3 min. After an additional wash in PBS, the cells were observed under Nikon ECLIPSE TS100 (Japan).

To further evaluate cell proliferation, an EdU assay was conducted using the BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 594 (Beyotime, Shanghai, China), and Hoechst 33342 was used for nuclear staining according to the manufacturer’s instructions. Briefly, cells were incubated with EdU staining buffer for 8 h and fixed with 4% polyformaldehyde, and the nuclei were stained with Hoechst. The stained cells were photographed under a microscope. Cells that stained both red and blue were considered EdU-positive cells. Random fields were selected and imaged. Cells were counted using ImageJ software. The ratio of EdU-positive cells to total cells was calculated as the cell proliferation rate. The experiment was repeated three times.

Bone marrow mesenchymal stem cells were seeded in a 12-well culture plate at a density of 2 × 10⁵ cells per well. When the cells reached 70–80% confluence, the EdU incorporation assay was performed according to the manufacturer’s protocol with a BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 594 (Beyotime Institute of Biotechnology, Haimen, China). The cells were visualized with a LeicaTCS SP5 X confocal microscope (Leica, Germany) and photographed.

The effect of DHPITO on the cell proliferation of CRC cells was approximated using a BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 594 (cat no. C0078S; Beyotime Institute of Biotechnology) while precisely following the manufacturer’s instructions. Briefly, cells were harvested, counted, seeded into the CellCarrier™-96-well plate (PerkinElmer, Inc., Waltham, USA) at a density of 1 × 10⁴ cells per well, and cultured overnight. Cells were treated with DHPITO for 24 h at concentrations of 0, 10, 20 or 40 μM; subsequently, the cells were incubated with 10 μM EdU working solution for 4 h at 37 °C. After fixation with 4% paraformaldehyde for 30 min at room temperature, the cells were blocked with QuickBlock™ Blocking Buffer for Immunol Staining (cat no. P0260; Beyotime Institute of Biotechnology, Shanghai, China) for 1 h at 37 °C. Thereafter, the cells were incubated in a click reaction buffer for EdU staining for 30 min in a dark environment before being stained with Hoechst 33342 for 10 min. Finally, the images were captured and approximated using an Operetta CLS High Content analytical system (PerkinElmer, Inc., Waltham, MA, USA).

Cells seeded in 12-well plates were cultured to 50% confluence and then transfected. Forty-eight hours after transfection with siRNAs or overexpression plasmid, the cells were fixed and stained with a BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 594 (Beyotime, China) in accordance with the instructions. A fluorescence microscope (Olympus, Japan) was used to capture three randomly selected fields to visualize the number of EdU-positive cells.