2.4. Total antioxidant capacity assay

The total antioxidant capacity assay provides on overall scan of cellular antioxidant status and is a measure of the amount of reactive oxygen species (ROS) scavenged by non-enzyme antioxidants such as thiols, vitamins, ascorbate, bilirubin, and albumin. A total antioxidant capacity assay kit (Sigma-Aldrich ^® MAK187) was used to measure reduction of Cu²⁺ to Cu⁺ to assess total antioxidant capacity of gill and DG tissues using trolox as an antioxidant standard. Tissue samples (about 25-45 mg) were homogenized on ice in a lysis buffer. Following centrifugation at 13,000 g for 10 min at 4 °C, samples were diluted 1:100 and mixed with the Cu²⁺ solution in a 1:1 ratio in a microplate. The plate was incubated for 90 min away from light. Antioxidant capacity was measured based on absorbance readings at 570 nm.