The level of mRNA for defensins was measured in total RNA preparation by quantitative real time PCR as described in Methods. Expression of all genes was normalised to the expression of the endogenous reference gene GAPDH. The expression value in control cells was used as the baseline. Means followed by the same letter are not significantly different. Detection of the hBD2 peptide in human airway epithelial cells by immunofluorescence To determine if defensin peptides were present in the airway epithelial cells exposed to A. fumigatus, the hBD2 peptide was detected by immunofluorescence. Analysis of the hBD9 peptide was not performed since anti-hBD9 antibodies were not available. A549 or 16HBE buy 10058-F4 cells were
cultured on cover slips, subsequently exposed to either SC, RC, HF, latex beads or treated with Il-1β for 18 h, and stained with polyclonal anti-hBD2 antibody as described in Methods. As shown in Figure 7A, hBD2 was detected in the cytoplasm of airway epithelial 16HBE cells exposed PF-01367338 supplier to any of the morphotypes of A. fumigatus, but generally not in the untreated control culture or in the cells exposed to the latex beads, except for several individual cells that contained some amount of defensin peptides. These click here findings are consistent with the inducible expression of hBD2. Staining revealed the punctuated distribution of peptides
in the cytoplasm with a concentration in the perinuclear region. It should be observed that the expression of the hBD2 peptide was not detected in each cell of the sample exposed to A. fumigatus. Quantification of the differences in the number of cells detected with anti-defensin-2 antibody showed that the number of stained cells in the untreated control culture was 8 ± 4%. The percentage of stained cells increased to 32 ± 4.6% after Il-1 β-treatment, to 17 ± 4.5% after exposure to RC, to 28 ± 5.2% after exposure to SC and to 20 ± 5.1% after exposure to HF, while exposure to the latex Ibrutinib clinical trial beads did not affect
defensin expression (9 ± 3.9%) (Figure 7B). Similar results were obtained with A549 cells (data not shown). 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.