G1914

OKF6/TERT2 monolayers were obtained by seeding 1 × 10⁶ cells per well of a 6 wells plate and incubating for 24 h at 37 °C in 5% CO₂. In the case of THP-1 cells, these were prepared using 1 × 106 cells per well of a 6 wells plate and were activated for 48 h as mentioned above. Bacteria were grown, washed once with phosphate buffered saline solution (PBS), suspended in PBS and added to OKF6/TERT2 and THP-1 cultures at a multiplicity of infection (MOI) of 100 approximately. The plates were centrifuged at 300× g (RCF) for 10 min to ensure the contact between the cell layer and the bacteria; after centrifugation, plates were incubated for 90 min at 37 °C in 5% CO₂ to allow for internalization of bacteria. Cells were then washed and incubated with fresh medium (KSFM and RPMI as appropriate) supplemented with gentamicin (G1914 Sigma-Aldrich^®, Gillingham, UK) and metronidazole (M3761 Sigma-Aldrich^®, Gillingham, UK), (300 µg/mL and 200 µg/mL respectively) for the post infection times defined (2 or 24 h).

THP-1 cells were seeded using 1 × 10⁶ cells per well using 6-well plates and activated for 48 h as mentioned above. Bacteria were washed with phosphate-buffered saline solution (PBS), suspended in PBS, and added to THP-1 cells plates using approximately 100 bacteria as multiplicity of infection (MOI). The plates, to achieve the contact between the cells and the bacteria, were centrifuged for 10 min at 300× g (RCF). Plates were incubated for 90 min at 37 °C in 5% CO₂ allowing for the internalization of bacteria in the macrophagic cells. Finally, cells were washed and incubated with fresh medium supplemented with metronidazole and gentamicin (G1914 Sigma-Aldrich^®, Gillingham, UK) (M3761 Sigma-Aldrich^®, Gillingham, UK), (200 μg/mL and 300 μg/mL, respectively) for 2 and 24 h defined as the post infection times.