L. monocytogenes entry into Hela cells: gentamicin protection assay

HeLa cells, that are only permissive for InlB-mediated entry because they lack E-cadherins (the receptor for InlA-mediated entry)³⁹, were grown as monolayers in 24-well plates. 4 h before infection, their culture medium was replaced with 500 µl of D-MEM without serum. Precultures of each Listeria strain were diluted and grown until they reached early stationary phase (OD600 of 2) in BHI media at 37 °C. Bacteria were washed with pre-warmed PBS, resuspended in pre-warmed D-MEM to achieve a MOI of 120 (for WT strains) or 70 (for prfA* strains), and 500 µl of inoculum was added to each well of the 24-well plates. Plates were centrifuged at 200 × g for 1 min and then incubated at 37 °C and 5% CO₂ for 1 h. The inoculum was washed away twice with PBS containing 40 μg/ml gentamicin, and then D-MEM containing 25 μg/ml gentamicin and 10% fetal bovine serum was added to each well. Infection was allowed to proceed for 1 h, after which cells were washed in PBS and then lysed in 500 µl of ice-cold sterile water. Colony forming units were counted out the inocula and cell lysates by serial dilutions and plating on BHI-agar plates. For each strain, three distinct wells were infected, and each was counted thrice independently. The experiment was performed thrice, and results of technical triplicates from a representative experiment were plotted (average ± standard deviation).