S3e flow cytometer

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.

M1 and M2 HMDMs and THP-1 macrophages were dissociated using enzyme-free cell dissociation buffer (13,151,014, Gibco) and scraping. Macrophages were stained with cell surface marker antibody or corresponding isotype control listed in Supplementary Table 1. Cells were resuspended in FACS buffer with 7-AAD viability dye (420,404, BioLegend) and incubated for 10 min at room temperature prior to data acquisition. Data were collected using an Attune NxT flow cytometer or Bio-Rad S3e flow cytometer and analysis was performed using Kaluza or FlowJo. Live macrophages were gated as 7-AAD negative cells. Delta median fluorescence intensity (MFI) was calculated as MFI of positive stained cells minus MFI of the isotype control stained cells.

M1 and M2 HMDMs and THP-1 macrophages were dissociated using enzyme-free cell dissociation buffer (13,151,014, Gibco) and scraping. Macrophages were stained with cell surface marker antibody or corresponding isotype control listed in Supplementary Table 1. Cells were resuspended in FACS buffer with 7-AAD viability dye (420,404, BioLegend) and incubated for 10 min at room temperature prior to data acquisition. Data were collected using an Attune NxT flow cytometer or Bio-Rad S3e flow cytometer and analysis was performed using Kaluza or FlowJo. Live macrophages were gated as 7-AAD negative cells. Delta median fluorescence intensity (MFI) was calculated as MFI of positive stained cells minus MFI of the isotype control stained cells.