Cfm 300

The quantification of cell proliferation was performed through EdU incorporation assay using BeyoClick™ EdU-594 Cell Proliferation Kit (Beyotime, Shanghai, China) according to the manufacturer's instruction. Particularly, cells were treated with 10 μM EdU and incubated for 2 h at 37°C. Then, the cells were fixed with 4% formaldehyde and permeabilized with 0.3% Triton X-100. Fixed cells were washed with PBS three times and incubated with Click Reaction Solution containing Azide 594 for 30 min in the dark before counterstaining nuclei with Hoechst 33342 (Beyotime). The EdU-positive rate of each group was assessed by selecting three random fields under a fluorescent microscope (CFM-300, Nikon, Japan) and measured using Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA); then the mean values were used for analysis.

To assess NC viability in the NC-rich NP explant model after culturing for 7 days, NC-rich NP explants were dyed with fluorogenic ester calcein-AM (CAM; Dojindo) to detect live cells, and with propidium iodide (PI; Sigma-Aldrich) to detect dead cells. The tissues were incubated with 2 mM CAM and 4.5 mM PI for 30 min at 37 °C in the dark and gently washed with PBS three times. A fluorescence microscope (CFM-300; Nikon) was used for image acquisition.