Standardizing Aspergillus fumigatus Conidia

A. fumigatus (ATCC^® 204305; Thermo Fisher Scientific, Waltham, MA, USA) was cultivated at 37 °C on malt agar (MEA) for 2 days, then harvested with 0.1% Tween 20 (Sigma Aldrich, St. Louis, MO, USA) in deionized water. The supernatant was filtered (Filcon, BD Biosciences, Franklin Lakes, NJ, USA), washed twice with cold Dulbecco’s phosphate-buffered saline (DPBS) (Thermo Fisher), and stored at 4 °C in DMEM. The concentration of the stock solution was calculated microscopically (40× magnification; Zeiss Axiocam, Carl Zeiss AG, Oberkochen, Germany) with a Neubauer counting chamber and by counting colony forming units (CFU) (40× magnification; Zeiss Axiocam). CFU plates on MEA were used to control the concentration. In our final standardization protocol before co-culture with NGs, Aspergillus conidia were pre-incubated at 37 °C for 3 h for maturation and swelling to stimulate the NG response through changes in surface proteins [42 (link)].