Mbs263555

For in vitro experiments, cells were treated with agents for 1 day. The medium was then exchanged to remove agents, and the cells were incubated for 1 additional day with the consistent basial medium. CM was subjected to low-speed centrifugation at 2000 rpm for 10 min. The cell-free supernatants were centrifuged at 4000 rpm for 10 min and subjected to filtration with a 0.22-μm polyethersulfone membrane (Sigma, St. Louis, MO, USA). The supernatants were further centrifuged at 10,000× g for 30 min at 4 °C to remove remaining cell debris and at 100,000× g (Type 90 Ti Rotor, Beckman, Brea, CA, USA) overnight at 4 °C to remove exosomes. For in vivo experiments, CM was harvested from the fetal bovine serum-free medium and treated by a filter with a cutoff molecular weight of 3 kDa. The levels of aggrecan and type II collagen in C28/I2 chondrocytes in response to 1 µg/mL of Hsp90ab1 were determined using ELISA kits (MBS262707 and MBS263555; MyBioSource).