Intracellular survival assay of S. aureus

For the intracellular bacterial survival assay, macrophages seeded in 24-well plates at 2.5×10⁵ cells/well were infected with S. aureus at an MOI of 10. After 1 h infection, cells were washed with PBS and extracellular bacteria were eliminated by the addition of 20 µg/ml gentamycin in cell culture medium. After 24 h incubation at 37°C, cells were rinsed with PBS and lysed in 1 ml ice-cold sterile water for 20 min at 4°C. Intracellular bacteria were diluted in 5-fold serial dilutions and then plated on Tryptic soy agar plates. Colony forming units (CFU) counts were determined 24 h after incubation at 37°C.