Sorvall legend rt

Fungal culture supernatants were prepared in flasks inoculated with 1 x 10⁶ A. fumigatus conidia/mL in 100 mL LB and incubated at 37 °C [23 (link)]. After 26 h, culture supernatants were filtered through either a 0.22 μm nylon membrane (Steritop^TM, Waltham, MA, USA) or Miracloth (Millipore Sigma, Burlington, ON, Canada). Culture supernatants were either lyophilized (Labconco, Kansas City, MO, USA) or stored at −20 °C.
Bacterial culture supernatants were prepared in flat-bottom, non-tissue, culture-treated, 24-well plates (Falcon, Burlington, VT, USA) inoculated with 2 mL of 9.0 × 10⁷ P. aeruginosa CFU/mL in LB per well with or without 0.5% L-arabinose (BioShop, Burlington, ON, Canada) and statically incubated at 37 °C. 48 h old culture supernatants were aspirated, separated from bacteria by centrifugation (Sorvall Legend RT+, Thermo Scientific, Waltham, MA, USA) at 2643× g for 30 min, and residual planktonic bacteria were heat-killed for 1 h at 60 °C [6 (link)]. Culture supernatants were dialyzed through 3.5 kDa molecular weight cut-off cellulose membrane (Fisherbrand, Pittsburgh, PA, USA or Spectrum Labs, Waltham, MA, USA) in 2 passes of deionized water, 1 pass of deionized water with 0.2% sodium azide (BioShop, Burlington, ON, Canada), 3 additional passes of deionized water, and 3 passes of 0.1% PBS. Culture supernatants were then either lyophilized or stored at 4 °C.

Fifteen bacterial cultures were used in this study; five Listeria monocytogenes, five Escherichia coli O157:H7, and five Salmonella enterica isolates. Details of each strain used can be found in Table 1. Bacterial cultures were preserved in Tryptic Soy broth (TSB, Bacto, Difco, Becton Dickinson, Sparks, MD) containing 30% glycerol and stored at −80°C until use. Cells were activated by three successive 24 hr transfers into TSB and incubated at 37°C. Activated cells were centrifuged (Sorvall Legend RT+, Thermo Scientific, Waltham, MA) at 2000g for 10 min at 22°C, the pellet resuspended in 0.1% buffered peptone water, and washed twice more to yield a bacterial cocktail of approximately 8.0 log CFU/ml. Cultures were diluted 10‐fold into sterile peptone water to yield a concentration of approximately 7.0 log CFU/ml. This dilution was used in the disk diffusion assays.

Corning Costar 7007 ultra-low attachment round bottom 96-well plates and phosphate buffered saline (PBS) were obtained from Corning (Corning, NY). Dulbecco’s Modified Eagle Medium (DMEM), penicillin-streptomycin, and HCS CellMask Deep Red stain were acquired from Thermo Fisher Scientific (Waltham, MA). Laminin-I was purchased from Trevigen (Gaithersburg, MD). For fixation, 8% paraformaldehyde (PFA) solution was obtained from Electron Microscopy Sciences (Hatfield, PA). Parafilm “M” was obtained from Bemis (Oshkosh, WI). Fetal bovine serum (FBS), Agar powder, and Triton X-100 were purchased from Sigma-Aldrich (St. Louis, MO). The High-Density Polyethylene (HDPE) inserts were acquired from Hero Glue (Las Vegas, NV). The neoprene gasket, absorbent mat, and nylon mesh for the containment base were obtained from McMaster-Carr (Elmhurst, IL). All other device components were fabricated with a Stratasys (Rehovot, Israel) Objet 30 Polyjet 3D printer using VeroWhitePlus acrylic photopolymer and support material. Centrifugation was conducted using a Sorvall Legend RT obtained from Thermo Fisher Scientific (Waltham, MA). For confocal microscopy, a Zeiss (Oberkochen, Germany) LSM 880 was used.