Figure 7
Localisation of the hBD2 peptide in epithelial bronchial 16HBE cells. 16HBE cells were seeded at 5 × 105 cells per well in 1 ml of DMEM/F12 on 18-mm-diameter cover slips in 12 well plates in triplicate and grown for 16 h at 37°C. After washing the cover slips with PBS-BSA, the cells were exposed to either latex beads, ethanol fixed conidia or ethanol fixed HF for 18 hours. Il-1β was used as a positive control. Following washing with PBS, the cells were fixed with a paraformaldehyde solution for 30 min at 37°C. The slides were then incubated in 1% BSA/PBS-Triton 0.05%, followed by a solution of 10% normal goat serum. After washing, polyclonal rabbit anti-human hBD2 at a dilution of 1:250 was applied as primary antibody overnight at 4°C, followed by incubation with FITC-labelled goat anti-rabbit secondary antibody at a dilution of 1:300 for 4 hours at room temperature. After washing, the cover slips were mounted on slides with ProLong antifade Vectashield. Samples were viewed with a Zeiss fluorescence microscope using ×400 magnification. The arrows indicate the cells stained with anti-hBD2 antibody. The percentage of stained cells was computed from triplicates of four experiments. Means followed by the same letter are not significantly different. +, presence; -, absence of Il-1β, A. fumigatus fixed organisms and latex beads. The punctuated localisation of the signal, which is concentrated adjacent to the nucleus (arrow), was observed. The data shown are representative of four independent experiments.