Phagocytosis assays.

Antibiotic protection assays were carried out using 200 μg/ml gentamicin as previously described (65). In brief, this involved incubating MDM or RAW cells with bacteria with or without antibiotics, washing the cells after 2 h, and then plating the lysate for bacterial counts to measure internal bacteria versus adherent plus internal bacteria. Intracellular survival experiments were performed using THP-1 cells, with MOI resulting in approximately equal uptake per THP-1 cell of TIGR4 or TIGR4 Δcps bacteria, followed by addition of gentamicin (200 μg/ml) after 30 min of incubation to kill extracellular bacteria and then addition of 2% saponin at the specified time points to lyse the cells for plating to obtain bacterial CFU. Microscopy phagocytosis assays were performed using FAM-SE-labeled bacteria opsonized in human serum, staining of external bacteria with phycoerythrin (PE)-labeled goat anti-human IgG (Sigma-Aldrich, Gillingham), and DAPI (4′,6-diamidino-2-phenylindole) staining of the nuclei, using a Hermes microscope for imaging (Biotech-Europe, Prague, Czech Republic). Images were analyzed on Metamorph (Metamorph, Inc.) software. Flow cytometry phagocytosis assays were performed using FAM-SE-labeled S. pneumoniae as previously described (59).