Intracellular MRSA Killing Assay

After 24 h stimulation with S. epidermidis, skin cells were washed twice with warmed plain IMDM and infected with MRSA at an MOI of 20 for 1 h. Cells were washed twice with IMDM after infection and fresh media containing gentamicin (50 μg/mL) was added for 30 min to eliminate extracellular bacteria. Samples were collected 30 and 90 min after intracellular infection for enumeration of intracellular colony forming units (CFU) and for FISH flow analysis as described before (43). To release intracellular bacterial load, cells were subjected to hypotonic lysis with 0.1% Triton X in PBS. Lysates were plated on agar plates containing 10 μg/mL chloramphenicol for CFU quantification (43).