Chondrocyte culture

Primary bovine chondrocytes were isolated from the metacarpophalangeal joint of cows (2–5 years old) obtained from a slaughterhouse within 3 hours post mortem. Chondrocytes were obtained by incubating cartilage slices overnight in Collagenase B (for details see [21]). Chondrocytes were seeded in DMEM/F12 1:1 containing 10% fetal calf serum (Thermo Scientific UK) at a density of 0.5 × 10⁵ cells per cm² in 6-well plates (Greiner Bio-one International, the Netherlands) for protein studies, or in 24-well plates (Bio-one International, the Netherlands) for mRNA experiments. Cells were cultured for 1 week at 37 °C and 5% CO₂ before the start of the experiments. For explant studies, four 7-mm² explants per condition were pooled in 1 ml of medium. Both monolayer and explant studies were repeated in different donors.