Fungal and Bacterial Supernatant Preparation

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.