Intracellular survival assay
The assays were performed in a 24-well tissue culture plates (500µl
THP-1-CWT cells per well). Bacterial cultures were washed twice with
sterile PBS and adjusted to reach a multiplicity of infection of 10:1.
Cells were infected and incubation time for phagocytosis was 1 h. The
cells were then washed once with HBSS, and the remaining extracellular
bacteria were killed by incubation with lysostaphin (2 μg/ml) and
gentamicin (200 μg/ml). After 1 h (t0) the cells were washed twice with
HBSS and then incubated in HBSS containing gentamicin (200 μg/ml) for 24
h (t24). At the indicated time points, the cells were washed once with
HBSS and incubated in 0.1% TritonX-100 for 5 min to disrupt the host
cells. Appropriate dilutions were plated on tryptic soy agar plates and
incubated at 37°C for the enumeration of colony forming units (CFU) on
the following day. The Inhibitor for acidification of the phagosome,
BafilomycinA1 (0.1 µM) (Sigma)
was added after 1 h of phagocytosis until indicated timepoints.