Development of a simple 96-well plate method for evaluation of antioxidant activity based on the oxidative haemolysis inhibition assay (OxHLIA)

OxHLIA is a cell-based antioxidant assay for evaluating inhibition of free radical-induced haemolysis in sheep erythrocytes by antioxidants. The original OxHLIA was performed in a test-tube format, and the degree of haemolysis was determined by absorbance of the supernatant after centrifugation. This made it difficult to evaluate large numbers of samples at the same time. Thus, we first showed here that the degree of haemolysis can be determined by turbidity of the erythrocyte suspension without centrifugation. We then developed OxHLIA in a 96-well plate format by adapting the turbidity method. There have already been some methodological studies on cell-based antioxidant assays using erythrocytes, and the method described here was comprised of the best mix of these earlier works with ingenious modifications. This plate method enables evaluation of large numbers of samples in small quantity at the same time with satisfactory precision and reproducibility.