Superoxide dismutase activity assay

We measured cartilage superoxide dismutase (SOD) activity spectrophotometrically in triplicate using a Superoxide Dismutase Activity Assay (Cayman Chemical) and a microplate reader (Tecan) following the manufacturer’s protocol. Two frozen explants per animal were cryopulverized and suspended in 400μL 50mm KPO4 pH 7.2. Sample homogenate was then pulse-sonicated 3×10 seconds on ice and cleared at 12,000 RCF. The supernatant was separated and incubated with water or 10mM KCN for 45 minutes. KCN selectively inhibits SOD1 and SOD3 allowing for the separate quantification of SOD2. The combined activity of SOD 1 and 3 was calculated as total SOD activity minus SOD2 activity.

We measured cartilage superoxide dismutase (SOD) activity spectrophotometrically using a Superoxide Dismutase Activity Assay (Cayman Chemical) following the manufacturer’s protocol. SOD2 activity was measured in the presence of KCN to distinguish from the cyanide-sensitive isoforms (i.e., SOD1 and SOD3). We determined the optimal KCN concentration by testing the inhibition of SOD activity with 3–30mM KCN, and we selected the lowest concentration (20mM) that provided maximal inhibition. Enzymatic activity was normalized to total protein content.