Fm 1 43fx lipophilic styryl dye

Bacteria were first stained with FM 1-43FX lipophilic styryl dye (Invitrogen F35355) (5 μg/mL) for 5 min to stain the outer membrane of the bacteria. The stained cells were spun down at 1000 g for 3 min, washed with media and resuspended in its original volume to be used for experiments. Normoxic and hypoxic pre-treated epithelial cells were infected with stained Fnn at 50:1 multiplicity of infection (MOI, Bacteria:Epithelial) for 1 or 4 h in their respective oxygen environments. Following infection, cells were washed twice with PBS, trypsinized, and collected for flow cytometry and cell sorting experiments. Cells were then loaded into an S3e flow cytometer (Bio-rad) and gated for single cells. 50,000 cells per sample were analyzed for green fluorescence due to intracellular F. nucleatum. Median fluorescence was determined using FlowJo10 before transferring data to GraphPad Prism for statistical analysis.

Live immunofluorescence visualization was achieved by staining epithelial cells according to manufacturer’s instructions with NucBlue (ThermoFisher Scientific 451 R37605) and Membrite 568/580 (Biotium BTM30095) for the cell’s nucleus and membrane respectively. F. nucleatum was stained with FM 1-43FX lipophilic styryl dye (Invitrogen F35355) (5 μg/mL) for 5 min to stain the outer membrane of the bacteria. Adhered epithelial cells were infected with F. nucleatum at 50:1 MOI for 4 h. Z-stack images were then taken with an LSM800 confocal microscope (Zeiss) to confirm intracellular invasion of bacteria. 60 slices within a total z height of 22 µm were obtained at a resolution of 1024 × 1024 pixels upon excitation with lasers 488 nm, 561 nm, and 405 nm. To confirm persistence of intracellular bacteria, z-stack images were again taken at 24 h and 3D structures were recreated using Zen Blue.