Conidial Labeling and Swelling in A. fumigatus

Wild-type A. fumigatus strain ATCC 46645 and the corresponding pigmentless pksP mutant (14 (link)) were cultivated on Aspergillus minimal medium (AMM) agar plates as described elsewhere (58 (link)). Conidia were grown for 5 days at 37°C and harvested with 0.9% (wt/vol) NaCl–0.01% (vol/vol) Tween 20. Conidia were labeled with calcofluor white (CFW; Sigma-Aldrich) or fluorescein isothiocyanate (FITC; Sigma-Aldrich) as previously described (13 (link)). To achieve swollen conidia, 4 × 10⁸A. fumigatus wild-type conidia were inoculated in 10 mL RPMI 1640 for 5 h at 200 rpm and 37°C. Subsequently, the conidial suspension was centrifuged at 1,000 × g for 5 min. The supernatant was discarded and the swollen conidia were resuspended in 3.7% formaldehyde in phosphate-buffered saline (PBS) and fixed for 30 min at 4°C. After fixation, the conidia were washed three times by centrifugation at 1,000 × g for 5 min and resuspension in PBS.