A. fumigatus CEA10 (46 (link)) and CEA10-RFP (47 ) strains were cultured on glucose minimal medium slants at 37 °C for 4–7 days prior to harvesting conidia for experimental use. To generate AF633-labeled or FLARE conidia for experimental use, 7×108 CEA10 (for AF633-labeled) or CEA10-RFP (for FLARE) conidia were rotated in 10 μg/ml Sulfo-NHS-LC-Biotin (Thermo Scientific) in 1 ml of 50 mM carbonate buffer (pH 8.3) for 2 hr at 4 °C, incubated with 20 μg/ml Streptavidin, Alexa Fluor™ 633 conjugate (Molecular Probes) at 37 °C for 1 h, resuspended in PBS and 0.025% Tween 20 for use within 24 hr (12 (link)).
To generate morphologically uniform heat-killed swollen conidia, 5×106/ml conidia were incubated at 37 °C for 14 hours in RPMI-1640 and 0.5 μg/ml voriconazole and heat-killed at 100 °C for 30 minutes (48 (link)). To infect mice with 3–6×107A. fumigatus cells, conidia were resuspended in PBS, 0.025% Tween-20 at a concentration of 0.6–1.2×109 cells/mL and 50 µl of cell suspension was administered via the intratracheal route to mice anesthetized by isoflurane inhalation.
To generate morphologically uniform heat-killed swollen conidia, 5×106/ml conidia were incubated at 37 °C for 14 hours in RPMI-1640 and 0.5 μg/ml voriconazole and heat-killed at 100 °C for 30 minutes (48 (link)). To infect mice with 3–6×107A. fumigatus cells, conidia were resuspended in PBS, 0.025% Tween-20 at a concentration of 0.6–1.2×109 cells/mL and 50 µl of cell suspension was administered via the intratracheal route to mice anesthetized by isoflurane inhalation.
