Hoechst 33342

Hoechst 33342 staining was used to study the apoptosis of ATDC5 cells. Cells were seeded into a 96-well plate at a density of 7×10³ cells/well, cultured in 100 µl medium and treated with HMGB1 (0, 10, 50, 100 and 300 ng/ml) or IL-1β (5 ng/ml) in a humidified incubator with 5% CO₂ at 37°C overnight. Subsequently, the medium was removed, and the cells were fixed at 4°C in 4% paraformaldehyde for 1 h and permeabilized in saponin (0.1% v/v in PBS-BSA). Hoechst 33342 (1 µg/ml; Promega Corporation) was added to each well, and the cells were further incubated in the dark for 30 min at 37°C. To assess specific apoptosis, apoptotic and living cells with condensed and fragmented nuclei were visualized using a blue filter of a Nikon TE2000-U inverted fluorescence microscope (Nikon Corporation) at ×200 magnification. The experiments were repeated three times.

PG (sodium salt, 17 and 41 kDa), deoxycytidine (dCyd), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide, and STZ were obtained from Sigma-Aldrich Corporation (St Louis, MO, USA). N,N-dimethylform-amide, dicyclohexylcarbodiimide, and dimethylaminopyridine (DMAP) were purchased from Fluka (Buchs, Switzerland). The compounds 2-mercaptoethanol, AEBSF, and Hoechst 33342 were obtained from Promega Cooperation (Madison, WI, USA), and 5-(aminoacetamido) fluorescein (fluoresceinyl glycine amide) (AF) was supplied by Invitrogen (Grand Island, NY, USA). The MicroScint™ 40 scintillation cocktail was obtained from PerkinElmer (Boston, MA, USA).
The [5-³H]-2′-deoxycytidine (³H-dCyd) was supplied by American Radiolabeled Chemicals, Inc. (St Louis, MO, USA). The Snakeskin™ dialysis tube (molecular weight cutoff =10,000 Da) was purchased from Pierce (Rockford, IL, USA). The Amicon Microcon centrifugal filter device (molecular weight cutoff =10,000 Da) was purchased from EMD Millipore (Billerica, MA, USA). All other solvents and reagents were commercially available and were of analytical or HPLC grade.