SLO-mediated hemolysis assay.

CSF obtained from mid-exponential-phase cultures was analyzed for SLO hemolytic activity (78, 79). Briefly, sterile CSFs were prepared from 10-ml cultures by centrifugation of the cultures at 3,220 × g for 10 min. Supernatant fluids were then filtered through a 0.2-μm nylon filter. Dithiothreitol (DTT) was added to 1 ml of CSF to a final concentration of 2 mM. Then, 50 μl of CSF was added to a 96-well plate with 50 μl of rabbit red blood cells (RBCs), which had been diluted in sterile PBS to a concentration of 5% (vol/vol). Plates were incubated at 37°C for 30 min and centrifuged at 3,220 × g for 5 min to pellet intact erythrocytes. Supernatants were transferred to a new 96-well plate, and extracellular hemoglobin from lysed RBCs was measured with A₅₄₀. To confirm that hemolysis was due to SLO, a neutralizing polyclonal SLO antibody (Abcam) was incubated with CSFs for 30 min at 37°C before the addition of erythrocytes. In addition, 100% hemolysis was defined with control wells containing 50 μl of RBCs that had previously been diluted to a concentration of 5% RBCs (vol/vol) in sterile water.