Invasion assay

Invasion was quantified by gentamicin protection assay [36,51,52]. HEp-2 cells were seeded into 24-well cell culture plates (~10⁵/well) and allowed to attach overnight at 37° C with 5% CO₂). Salmonella strains were grown overnight without aeration in LB buffered with 100 mM HEPES, pH 8.0, with antibiotics and fatty acids or control solvent. Overnight cultures were washed with sterile PBS. Bacteria were added to the HEp-2 cell monolayer (MOI 1:10), centrifuged briefly (10 minutes at 200 x g) and incubated for 1 hour at 37°C. Wells were washed three times with sterile PBS followed by addition of media with 200 μg/mL gentamicin and incubated for 1 hour. Cells were then washed three times with sterile PBS and lysed by 0.1% Triton X-100. Intracellular bacteria were enumerated by spreading lysates onto LB agar plates with appropriate selection.