Fcs express version 7

Cells were plated on 18 mm coverslips in a 12-well plate (Warner Instruments Corporation, Hamden, CT, USA) at a density of 250,000 cells in 1 mL of complete medium for microscopy experiments and on 6-well plates at a density of 500,000 cells per well in 2 mL of complete medium for flow cytometry experiments. Cells were allowed to recover for 24 h and then were exposed to AgNPs or Ag⁺ for 24 h at 37 °C. Medium was removed and fresh media containing 10 µM of the lipid peroxide specific dye, Liperfluo (Dojindo Molecular Technologies, Rockville, MD,) was added for 30 min. Cells were then washed twice with PBS and fluorescence was measured using an Olympus FV1200 spectral laser scanning confocal microscope and a FACS Canto II Analyzer (BD Biosciences). Analysis of the data was performed using FCS express version 7 (De Novo Software, Glendale, CA, USA).

Cells were plated on 6-well plates at a density of 500,000 cells per well in 2 mL of complete medium. Cells were allowed to recover for 24 h and then were exposed to AgNPs or Ag⁺ for 24 h at 37 C. Medium was removed and fresh media containing 50 µM of DCP-NEt₂-Coumarin (DCP-NEt₂C) prepared as previously described [35 ] was added for 30 min. Cells were then washed twice with PBS, fixed with 100% methanol and fluorescence was measured using a FACS Canto II Analyzer (BD Biosciences). Analysis of the data was performed using FCS express version 7 (De Novo Software, Glendale, CA, USA). To control for any potential contribution to the fluorescence profile due to AgNPs themselves, unstained AgNP treated and untreated cells were prepared and analyzed as described above. As shown in Additional file 1: Fig. S1A, the fluorescence profiles overlapped, indicating no difference between the two groups, and the magnitude of fluorescence was less than 0.5% of the DCP-NEt₂C stained, untreated controls. Because these background measurements fell within the standard deviation of normal sample variation, any contribution of AgNP absorbance was deemed negligible to the overall results.

Cells were plated at a density of 500,000 cells per well on 6-well plates in 2 mL of complete medium and allowed to adhere for 24 h. Cells were exposed to AgNPs for 24 h. Afterward, fresh media containing 50 µM of DCP-NEt₂-Coumarin (DCP-NEt₂C) was added for 30 minutes. Cells were then washed twice with PBS, fixed with 100% methanol and fluorescence was measured using a FACS Canto II Analyzer (BD Biosciences). Analysis of the data was performed using FCS express version 7 (De Novo Software, Glendale, CA, USA).

Total 1x10⁶ cells of iDC or 27DC were washed three times with ice-cold Dulbecco’s PBS (Thermo Fisher Scientific) in the presence of BSA, and then blocked using Fc Receptor Blocker (Innovex Biosciences, Richmond, CA, USA) for 30 minutes at room temperature in the dark. Cells were washed twice in 2% BSA (MilliporeSigma, St. Louis, MO, USA) with 0.5% NaN3 (MilliporeSigma) in Dulbecco’s PBS (DPBS-BSA-NaN₃). Cells were then stained, including paired isotype controls, for 15 minutes at room temperature in the dark. The following antibodies, and their respective fluorochrome, were used: CD4 (Fluorochrome BV421; BioLegend, San Diego, CA, USA; Ca# 317434) and isotype control (Fluorochrome BV421; BioLegend; Ca# 400158), CCR5 (Fluorochrome APC-Cy7; BD Biosciences, Franklin Lakes, NJ, USA; Ca# 557755) and isotype control (Fluorochrome APC-Cy7; BioLegend; Ca# 400128), CXCR4 (Fluorochrome PE; BioLegend; Ca# 306506) and isotype control (Fluorochrome PE; BioLegend; Ca# 400114). The cells were washed twice in DPBS-BSA-NaN₃ and then run immediately on an LSR Fortessa flow cytometer (BD Biosciences). The results were analyzed using FCS Express version 7 (DeNovo Software, Pasadena, CA, USA).

Culturable S. sonnei isolates corresponding to genomes (n = 159) were grown overnight in 96 well flat bottom plates (Greiner Bio One, UK) containing 150 μl of TSB and diluted 1:100 (v/v) into fresh TSB and grown for 2 h. E. coli was grown and diluted into fresh media as was done for initial killing assays and 880 μl of mid-log phase culture was diluted in 52 ml of TSB and 130 μl of that was distributed into each well of the 96-well plate. 20 μl of the mid-log phase S. sonnei cultures were then added to each well to make up a final volume of 150 μl with 1:10 (E. coli: S. sonnei) of the competition mixture and incubated overnight. The overnight competition mixtures were then diluted and GFP expressing E. coli cells which had survived the competition with S. sonnei were counted using a Bio-Rad ZE5 Cell Analyzer in a total of 10,000 events per well. The percentage of GFP expressing cells were calculated using FCS Express version 7 (De Novo Software) and plotted onto the phylogenetic tree using iTOL.⁴⁷ (link)