Phalloidin 650

For CLSM analysis, 0.5 × 10⁶ ds Red A. fumigatus conidia were applied to the apical surface and at several time points post infection as indicated in the Figure legends or Results, inocula were removed by gentle suction and the well was fixed using 4% paraformaldehyde solution for 10 minutes. After fixation the cells were treated as described above except staining the slides for mucin using rabbit anti-human MUC5B antibody (1:80; Abcam), followed by donkey anti-rabbit IgG (1:50 Biolegend). Again, actin localized to tight junctions was identified by using phalloidin-650 (1:20; Cell Signaling Technology). Cilia were stained using an anti-tubulin antibody (1:10; BD Pharmingen), and nuclei using Hoechst 33342 (Cell Signaling Technology). Images were captured using a Leica SP5 confocal laser scanning microscope and analysed using the LAS AF Lite or Imaris software. For each condition at least 50 cells were analyzed and used for quantifications.

For dual co-culture experiments, SAE cells were grown on collagen-coated Transwell inserts under perfused conditions as described and subsequently 2.5 × 10⁵ macrophages/well were allowed to adhere to the apical surface of the insert for two hours. Prior to adherence, macrophages were stained using 2.5 µM CFSE for detection by confocal microscopic analyses. After 2 h adherence, the medium from the apical surface was gently removed and the co-cultures were infected with 0.5 × 10⁶ dsRed A. fumigatus conidia to mimic fungal infection of the lower respiratory tract. 20 µl of the conidial suspension were added to the apical side of the co-cultures and incubated at 37 °C for various time points (1 h to 24 h dependent on the experimental set up). Inocula were removed by gentle suction and fixed with 4% paraformaldehyde solution for 10 minutes. After fixation the cells were washed three times with D-PBS solution, permeabilised for 15 minutes (eBioscience) followed by blocking with 1% BSA. Actin localized to tight junctions was identified by using phalloidin-650 (1:20; Cell Signaling Technology) and nuclei were stained using Hoechst 33342 (Cell Signaling Technology). Images were captured using a Leica SP5 confocal laser scanning microscope and analysed using the LAS AF Lite or Imaris software.

For dual co-culture experiments, NHBE cells were grown on collagen-coated Transwell inserts under perfused conditions as described and subsequently 2.5 × 10⁵ DCs/Transwell were allowed to adhere to the basolateral surface of the insert for one hour. Prior to adherence, DCs were stained using 2.5 µM CFSE for detection by confocal microscopic analyses. After 1hr adherence, the inserts were reverted back to their original position and the co-cultures were infected with 0.5 × 10⁶ dsRed A. fumigatus conidia to mimic fungal infection of the upper respiratory tract. 20 µl of the conidial suspension were added to the apical side of the co-cultures and incubated at 37 °C for various time points (30 min to 24 h dependent on the experimental set up). Inocula were removed by gentle suction and fixed with 4% paraformaldehyde solution for 10 minutes. After fixation the cells were washed three times with D-PBS solution, permeabilised for 15 minutes (eBioscience) followed by blocking with 1% BSA. Actin localized to tight junctions was identified by using phalloidin-650 (1:20; Cell Signaling Technology) and nuclei were stained using Hoechst 33342 (Cell Signaling Technology). Images were captured using a Leica SP5 confocal laser scanning microscope and analysed using the LAS AF Lite software (Leica).