Hoechst 33 258 dye

For flow cytometry analysis, cells were seeded onto 6-well plates at a density of 3 × 10⁵ cells per well overnight. SA-treated cells were dyed with Annexin V-FITC/PI apoptosis detection kit (KeyGEN BioTECH, Nanjing, China), and analyzed by the system of FACSAria flow cytometer (BD Biosciences, New Jersey, USA), and FlowJo software. For Hoechst 33,258 staining, cells were seeded in 6-well plates at 60–70% confluency, treated with SA, and stained with Hoechst 33,258 dye for 10 min at room temperature before examination (KeyGEN BioTECH, Nanjing, China).

After harvesting, the constructs were weighed and then digested for 24 h in 1 mL of 0.1 M PBS containing 2.5 U of papain (Beyotime, China), 5 mM cysteine-HCl and 5 mM Na2EDTA at 60 °C. The cell number was assessed based on the DNA content using Hoechst 33258 dye (KeyGen Biotechnology Nanjing, China), as previously described [21 (link)]. Hoechst 33258 is a fluorescent nucleic acid stain used to quantitate double-stranded DNA in solution. The fluorescence of the samples was converted to cell numbers using a cell-number standard curve created previously. The glycosaminoglycan (GAG) content was determined using the 1, 9-dimethylmethylene blue (DMMB; Jianglaibio, China) dye-binding assay and a shark cartilage chondroitin sulfate reference sample (C4348; Sigma, USA). The GAG content was normalized to the cell number to assess the single-cell biosynthetic activity.