Cck 8 reagent

Based on the CCK-8 assay and 5-ethynyl-2’-deoxyuridine (EdU) incorporation assay, cell proliferation was quantified. For the CCK-8 assay, the transfected A549 cells were seeded on 96-well plates (1 × 10³/well) with 10 μl of CCK-8 reagent (FC101, TransGen, Beijing, China) and counted every 24 h for three days. Counting was carried out after cell attachment for 24 h (labeled 24 h). For the EdU incorporation assay, cell viability was estimated using EdU reagent (C0075s, Beyotime, China) following the manufacturer’s instructions. Briefly, the transfected A549 cells were incubated with EdU reagent for 2 h and processed. Then, the cells were fixed with 4% paraformaldehyde, fluorescent dye was used to stain cells, and the cells were visualized under a fluorescence microscope (Nikon laser confocal microscope, Nikon, Japan).

Infected COLO205 and SW480 cells were seeded in 96-well plates at a density of 1 × 10⁴ cells/well for 24 h. After treatment with NP (10^–6 M) or DMSO (n = 6 per group) for 24 h, 10 μL of cell counting kit-8 assay (CCK8) reagent (TransGen, Beijing, China) was added to the cells, after which they were incubated for 4 h in an incubator at 37°C with 5% CO₂. Absorbance was measured at 450 nm using a microplate analyzer.

CCK-8 assay was performed to determine the number of living cells during cell proliferation. The HepG2 cells were inoculated in 96-well plates at a density of 5 × 10³/well. After transfection for 24, 48, 72 or 96 h, 10 μL CCK-8 reagent (Transgen Biotech Co., LTD) was added to each well and incubated at 37 ℃ for 1 h. Measure the absorbance at 450 nm according to the instructions.
EdU (5-Ethynyl-2′-deoxyuridine) assay was performed to detect HepG2 cells in the dividing phase (S phase) and analyze HepG2 cell proliferation using BeyoClick^TMEdU-488 Cell Proliferation Kit (Beyotime). The density of HepG2 cells was adjusted, and 5 × 10³ per well were spread in a 24-well plate. After transfection for 24 h, EDU reagent was mixed into the culture medium for 12 h. After fluorescence staining, the cell density was observed under a fluorescence microscope.

Cell proliferation assays were carried out using the cell counting kit‐8 (CCK‐8) assay and colony formation assay. For CCK‐8 assay, cells were seeded in 96‐well plates at a density of 5 × 10³ cells per well and incubated at 37 °C in 5% CO₂. An aliquot of 10%(v/v) CCK‐8 reagent (TransGen Biotech) was added to each well and incubated at 37 °C for 2 h. The absorbance (A) was measured at the wavelength of 450 nm according to the manufacturer's protocol (Molecular Devices LLC, Sunnyvale, CA, USA). Samples were then measured every 24 h after 4 days of culture. The experiments were repeated independently for five times. For Colony formation assay, cells were seeded in six‐well plates at a density of 5 × 10² cells per well and incubated at 37 °C in 5% CO₂ for 2 weeks. After that, formed colonies were fixed with 4% paraformaldehyde (Beyotime Biotechnology, Shanghai, China) for 20 min and then stained with 0.1% crystal violet (Beyotime Biotechnology) for 10 min. A number of clones were imaged and counted under microscope. The experiments were repeated independently three times.

The cells (U‐CH1 and U‐CH2) were seeded on 96‐well plates (1 × 10³/well). After 24 h of culture, 10 µL of CCK‐8 reagent (FC101, TransGen) was added to the cells, and their viability was assessed by measuring the absorbance at OD450 at 12, 24, 48 and 72 h.

After a 9-day cultivation, the concentration of the 2 types of MSCs was adjusted to 2 × 10⁴ cells/mL, and these cell solutions inoculated in a 96-well cell culture plate with 100 μL/well. After culture for 1, 2, 3, and 4 days, 10 μL CCK-8 reagent (FC101-03, TransGen Biotech, Beijing, China) was added to each well, and the plates were incubated at 37 °C with 5% CO₂ for 2 h. The OD values were determined at 450 nm with a microplate reader (Bio-Rad, USA). The cell proliferation rate was calculated as follows: rate (day X) = [OD450 (day X)-OD450 (day X-1)]/OD450 (day X-1). The sample size was 6.