Beyoclick edu 555 cell proliferation kit

The EdU assay was conducted to investigate the impact of arachidonic acid on the proliferation of NPC cell lines. HK1, CNE-2, and 5–8F cells were seeded in a 24-well plate at a density of 2 × 10⁵ cells per well and allowed to incubate overnight. Subsequently, a conditioned medium containing indomethacin at various IC50 concentrations (450, 300, 250 µg/mL) was applied for 24 h at 37 °C with 5% CO2. Following this, 100 µL of EdU (50 µM) was introduced to the culture medium for an 8-h incubation period. The cells were then fixed with 4% paraformaldehyde for 20 min, permeabilized using Triton X-100 to breach the nuclear membrane, and blocked with PBS for an additional 1 h at 25 °C. Finally, cell staining was performed using the BeyoClick™ EdU-555 Cell Proliferation Kit (Beyotime, China) in accordance with the manufacturer’s instructions. A negative control was established using 1% DMSO.

The BeyoClick™ EdU-555 Cell Proliferation Kit (Beyotime, China) was utilized following the instructions provided. Following 2-hour incubation with EdU, the cells were fixed with a 4% paraformaldehyde solution, permeabilized, and then stained with Alexa Fluor 555 and Hoechst 3342. Subsequently, the cells were examined using an inverted fluorescence microscope to evaluate the presence of EdU.

BeyoClick™ EdU‐555 Cell Proliferation Kit (Beyotime, China) was used after following the manufacturer's protocol to determine cell proliferation. A total of 3 × 10⁴ cells were cultured for 24 h in 96‐well plates. After a 2‐ h incubation with 20 μM 5‐ethynyl‐2'‐deoxyuridine (EdU) solution, both cell lines were fixed with 4% paraformaldehyde then secured using Apollo Dye Solution and DAPI. Cells were then photographed and counted under a C2+ confocal microscope (Nikon, Japan). The assay was carried out three times.

A BeyoClick™ EdU-555 Cell Proliferation Kit (Beyotime Bio, Shanghai, China) was used for the cell proliferation assay. The PASMCs were incubated with10 μmol/L EdU for 2 h at 37 °C after treatment. The cells were permeabilized with Enhanced Immunostaining Permeabilization Buffer for 15 min after fixation with 4% formaldehyde for 15 min at room temperature. After three washes with PBS, the cells were incubated with click additive solution for 30 min. PASMC nuclei stained with Hoechst-33,342 were used for the cell counts and examined using fluorescence microscope.

Caki-1 cells were stained using BeyoClick™ EdU-555 Cell Proliferation Kit (Beyotime, Shanghai, China). To be specific, Caki-1 cells (1.0×10⁵ cells/well) were seeded in a 6-well plate, transfected with NC or si-FDX1, and cultured in an incubator at 37°C for 72 h. Then, Caki-1 cells were incubated with EdU for 2 h, fixed with 1 mL paraformaldehyde (4%) for 15 min, and permeabilized with 0.3% Triton X-100 (Beyotime) for 15 min. After that, the Caki-1 cells were incubated with 500µL of the click reaction mixture for 30 min in the dark, washed three times with PBS containing 3% BSA, and incubated with Hoechst 33342 for another 10 min. Finally, fluorescence microscopy was used for detection.

VSMCs were seeded on coverslips in a 24-well plate before SCAP siRNA transfection. Following the instructions for the BeyoClick™ EdU-555 Cell Proliferation Kit (C0075S, Beyotime, Nanjing, China), -ve CTRi VSMCs and SCAPi VSMCs were separately cocultured with 20 μM EdU working solution away from light for 2 h at 37 °C. After that, the cells were fixed in 4% paraformaldehyde and permeabilized with 0.3% Triton X-100. Next, the samples were incubated with Click Reaction Buffer and counterstained with DAPI. Fluorescence images were captured with a Leica laser scanning confocal microscope (Weztlar, Germany). The number of EdU-positive cells was quantitated by ImageJ software.