Then the co-cultured cells were rigorously washed twice with phosphate buffered saline (PBS) to remove bacteria in suspension, trypsinized to detach mammalian host cells for flow cytometry , fixed with PBS supplemented with 1% formaldehyde, 0.33% BGS and 0.001% of sodium pyruvate and assayed for their GFP signals by flow cytome-try (FACSCanto II, Becton Dickinson).

(B) Flow cytometry of bacterial GFP associated with mammalian cells.

(D) Flow cytometry detection of surface Bl-integrins.

Flow cytometry data for GFP expression.

After co-culture of increasing amounts of GFP-expressing E. coli harboring a plasmid permitting arabinose-inducible control of invasin (pBACr-Aralnv) with HeLa cells, flow cytometry was conducted to obtain data shown in Fig 3A.

To simplify the data for comparison, a centroid and mean level of Bl-integrins at a given fraction of infected host cells were computationally estimated from each flow cytometry sample at different MOIs (Fig 3B).

This property was consistent with flow cytometry measurements (Fig 1B): At intermediate bacterial concentrations, a bimodal distribution of GFP fluorescence arises where within a single population there exist both uninfected cells (i.e.