Edu cell proliferation kit with alexa fluor 594

The cell proliferation was analyzed using a 5-Ethynyl-2′-Deoxyuridine (EdU) assay using the EdU Cell Proliferation Kit with Alexa Fluor 594 (Beyotime, #C0078S) to detect cell viability. First, the cells were seeded to a 6-well plate at 1 × 10⁶ cells per well and then 10 μM EdU was added in each well at 37 °C for 3 h. After being rinsed twice, the cells were prepared as a suspension and spread on a slide, and then fixed in 4% paraformaldehyde for 15 min Next, the slides were covered with a click reaction solution for 30 min in the dark. Subsequently, the cells were treated using 0.5% Triton X-100 for 15 min, and DAPI was applied to label the cell nuclei for 30 min. Cell proliferation was evaluated by using the ratio of EdU-stained cells (with red fluorescence) to DAPI-stained cells (with blue fluorescence). Images were analyzed using a fluorescence microscope (Nikon, Tokyo, Japan) at 100× magnification.

B-PE films were placed into a 48-well plate and were sterilized with ultraviolet light for 30 min. NIH3T3 fibroblasts and human vascular ECs were seeded into the plates at a density of 1 × 10⁴ cells/cm⁻², and were incubated in standard culture conditions for 24 h. The cell viability on the B-PE films was examined after 24 h seeding using Live/Dead staining, a two-color discrimination of living cells from dead cells. Living cells were stained with fluorescein diacetate (FDA), and dead cells were stained with propidium iodide. The fluorescent images were taken with a fluorescent microscope (IX81, Olympus). EdU Cell Proliferation Kit with Alexa Fluor 594 (Beyotime, Cat. No: C0078S) was used to visualize the proliferating cells. At 22-h post-seeding, 10 µM EdU was added into the medium. After another continuous culture of 2 h, the cells were fixed and stained for EdU uptake. Nuclei were counter-stained with Hoechst 33342. After an additional wash in PBS, the cells were observed under an inverted fluorescent microscope (DMI8, Leica).

10% (w/v) DEFs was prepared using DMEM/F12 medium with 10% (v/v) FBS according to required amount, the suspension was then vortexed vigorously for 5 min and centrifuged for 10 min at 3500 rpm. The supernatant was extracted for further cell culture experiments. Cell proliferation was determined by the uptake of 5‐ethynyl‐2‐deoxyuridine (EdU) into DNA using an EdU Cell Proliferation Kit with Alexa Fluor 594 (Beyotime, Shanghai, China) according to the manufacturer's instructions. Cells were seeded in 96‐well plates and incubated with the DEFs supernatant at 37 °C in a humidified 5% CO₂ incubator after 24 h. EdU (final concentration 10 µm) was added at the 12th hour of coculture, and the incubation continued for 12 h. Cells were further fixed, permeabilized, blocked and stained according to the instructions. Images were captured by fluorescence microscopy (LSM800, Zeiss, Germany) and analyzed by ImageJ software. Cell viability was determined by a CCK‐8 assay kit (Dojindo, Shanghai, China). The optical density of each well at 450 nm was measured using a microplate reader (Thermo Fisher) after HESCs coculture with DEFs supernatant for 24, 48, and 72 h.

Cell proliferation assay was performed using EdU Cell Proliferation Kit with Alexa Fluor 594 (Beyotime, Shanghai, China) and following the protocol described by the manufacturer. Cells were seeded onto a 24-well plate with coverslips, with 1x10⁵ cells/per well. Anticarin β (0.5 μM) was added and the control wells were treated with the same volume of PBS. Cells were treated for 12 h at 37˚C in a humidified incubator. Then, cells were treated with EdU (10 µM) for 2 h at 37˚C in a humidified incubator with 5% CO₂. Following incubation, the cells were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 in PBS containing 3% BSA (PBS - BSA) for 20 min. Then, the cells were immersed in Click reaction mixture for 30 min and in Hoechst 33324 (10 mg/mL; diluted 1:1,000 in PBS) for 10 min at room temperature protected from light. Before the observation, the cells were washed twice with PBS and mounted onto a glass slide. Images were obtained using a fluorescent microscope at the magnifications 20x.

The C2C12 myoblast cells were cultured in 96 wells and treated with Ad‐GFP or Ad‐NLS‐PGC‐1α4 for 24 h and analyzed with proliferative capacity with CCK‐8 assay (Beyotime, C0043) or EdU Cell Proliferation Kit with Alexa Fluor 594 (Beyotime, C0078S) following manufacturers' instructions.

HT-22 cell proliferation was determined by EdU incorporation assay via EdU cell proliferation Kit with Alexa Fluor 594 following the manufacturer’s instructions (Beyotime, China). HT-22 cells were seeded in six-well plates and were allowed to be treated with various drugs for 24 h. Cells were then incubated with 10 μM EdU solution in DMEM medium for 4 h. The cells were washed with washing buffer (PBS containing 3% BSA), followed by fixation of 4% polyformaldehyde for 15 min and then permeabilization with PBS containing 0.3% Triton X-100 for 20 min. After three times washing, cells were incubated with azide-conjugated Alexa Fluor 594 for 30 min in click addictive reactive buffer with 4 mM CuSO₄. Cells were then washed three times with washing buffer. DAPI (1:1,000, Beyotime, China) was incubated with cells in PBS solution for 10 min at room temperature. The cells in six different areas of each well were photographed under a fluorescent microscope and analyzed with ImageJ software. The percentage of proliferated cells was calculated as EdU-positive cell number/total cell number × 100%.

The proliferation ability of BCa cells was detected by EdU assay using the EdU Cell Proliferation Kit with Alexa Fluor 594 (Beyotime, China). Cells were washed with PBS three times and incubated with complete medium with 10 μM EdU in a cell incubator for at least 2 h. Then, the cells were washed with PBS to remove the EdU probe and culture medium, fixed with 4% paraformaldehyde for 10 min at room temperature, and stained with DAPI for 5 min in the dark. The cells were observed under a positive fluorescence microscope (Olympus, Tokyo, Japan) at 100x magnification in five random fields. The staining signals were captured, analyzed, and shown as fold changes compared with the control. The experiment was repeated three times.