Hemolysis assay

S. aureus strains 8325-4 were cultured in TSB at 37 °C until the post-exponential growth phase was reached (OD_600 nm = 2.5) in the presence of indicated concentrations of CUR. Following centrifugation (10,000 × g, room temperature, 5 min), 100 μl of bacterial culture supernatants were mixed with 900 μl PBS buffer containing 3.0% defibrinated rabbit erythrocytes and the mixtures were incubated at 37 °C for 20 min. After centrifugation (5,500 × g, room temperature, 1 min), the absorption at 543 nm of supernatants was measured and the hemolysis percentage was calculated relative to the control culture, which was regarded as having 100% hemolytic activity.

To investigate the effect of CUR on hemolysis induced by wild type (WT)-Hla or its mutants, 100 μl of purified Hla (500 ng/ml) was pre-incubated in microtiter plates with graded concentrations of CUR at 37 °C for 15 min. Then, 100 μl (5 × 10⁶cells/ml) of defibrinated rabbit erythrocytes was added to each well for another incubation at 37 °C for 20 min. Lysis of the cells was determined as described above.