Cultivation of A. fumigatus strains CEA10 (Fungal Genetics Stock Center; A1163), CEA17 ΔakuBku80 ΔpksP (48 (link)), and AfS35/pJW103 (36 (link)) was performed on malt agar plates (Sigma-Aldrich) supplemented to a final concentration of 2% (wt/vol) agar for 5 days at 37°C. Conidia were harvested in sterile, ultrafiltrated water filtered through a 30-μm pore filter (MACS, Miltenyi Biotec). Prior to confrontation with PLB-985 cells, conidia were opsonized with normal human serum (Merck Millipore). Briefly, 900 μl of spore suspension was mixed with 100 μl of normal human serum and incubated in a thermomixer at 37°C for 30 min shaking at 500 rpm. The spore suspension was washed three times by collecting spores via centrifugation at 1,800 × g for 1 min at 4°C and resuspending in fresh phosphate-buffered saline (PBS). Following washing, spores were enumerated in a Thoma chamber in preparation for infection assays.
Malt agar plates
Manufactured by Merck Group
Sourced in Switzerland
Malt agar plates are a type of laboratory culture medium used for the growth and isolation of various microorganisms, particularly fungi and yeasts. They provide a nutrient-rich environment that supports the cultivation of these organisms.
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2 protocols using malt agar plates
1
Cultivation and Opsonization of A. fumigatus Strains
2
Yeast Strain Cultivation and IFC Measurement
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Yeast strain Scc ATCC 9080 was obtained from DMSZ GmbH (Braunschweig, Germany).
Cells were first cultured overnight on malt agar plates (Sigma Aldrich, Buchs, Switzerland) at 30 °C, and then single colonies were picked and incubated in YPD liquid medium (Sigma) at 30 °C with rotary shaking at 200 rpm for maximally 24 h or as indicated in the text. A sample from the liquid culture at t = 23 h (exponential growth phase) was split into two, and one half was heat-treated at 70 °C for 15 min using a heat block for Eppendorf tubes. For IFC measurements, samples were diluted 1:20 in either (Fig. 1) the isosmotic IFC buffer AF6 (Amphasys, Switzerland) or (all other data) in 0.5x phosphate-buffered saline (PBS; Dulbecco's PBS, Sigma) and filtered using a 20 μm filter (Sysmex CellTrics).
Cells were first cultured overnight on malt agar plates (Sigma Aldrich, Buchs, Switzerland) at 30 °C, and then single colonies were picked and incubated in YPD liquid medium (Sigma) at 30 °C with rotary shaking at 200 rpm for maximally 24 h or as indicated in the text. A sample from the liquid culture at t = 23 h (exponential growth phase) was split into two, and one half was heat-treated at 70 °C for 15 min using a heat block for Eppendorf tubes. For IFC measurements, samples were diluted 1:20 in either (Fig. 1) the isosmotic IFC buffer AF6 (Amphasys, Switzerland) or (all other data) in 0.5x phosphate-buffered saline (PBS; Dulbecco's PBS, Sigma) and filtered using a 20 μm filter (Sysmex CellTrics).
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