Figures & data
Figure 1. Metabolic activity and viability measurements during cultivation of human PCLS. At different time points during cultivation of PCLS, the metabolic activity was measured using a WST-1 assay (A) and the viability was measured using an LDH assay (B). Each data point represents the mean ± SD of 4 PCLS from 5 to 8 lungs.
Figure 2. Measurement of metabolic activity and viability in LPS- and Triton X-100 -treated PCLS. On days 2 or 3, 7, and 12 or 13 of cultivation, PCLS were treated with different concentrations of LPS or medium only for 18 hours. As a control, PCLS were treated with 0.25 - 0.3 mM Triton X-100 for 1 hour. Metabolic activity was measured by a WST-1 assay. Each symbol on the graph represents PCLS from a given donor with the lines representing the mean value for the PCLS treated with each condition.
Figure 3. Representative images of human PCLS stained with Calcein AM and Ethd-1 at different days of cultivation. PCLS were treated with 2 concentrations of LPS (10 or 100 ng/mL), 2 concentrations of Triton X-100 (0.125 or 0.3 mM) or medium alone. PCLS were stained by Calcein AM and Ethd-1 and evaluated by confocal microscopy. Three stacks of 31 pictures (30-µm thickness) per slice were randomly selected on days 3, 7 and 13. The assay was conducted in duplicate.
Figure 4. Histology of lung airways in sections of PCLS. Images of airway epithelium from PCLS treated with medium only (control) or 100 ng/mL of LPS for 18 hours. Comparable PCLS were also treated with 0.25 mM Triton X-100 for one hour. Experiments were performed on days 3, 7 and 13 of cultivation. SM = smooth muscle, C = cilia, AE = airway epithelium. Original magnification 400 x.
Table 1A. Secretion of cytokines and chemokines by PCLS in response to LPS.
Table 1B. Level of COX-2 expression in PCLS stimulated by LPS.
Figure 5. Level of cytokines in culture supernatants from restimulated PCLS. PCLS were treated on day 2 of cultivation with either LPS, PMA/I, the 2010–2011 seasonal influenza vaccine (Flu) or tetanus toxoid (Tet) for 24 hours. After incubation, the level of cytokine in the culture supernatant was measured using the MSD Multi-Spot assay system: A) IFN-γ, B) TNF-α, C) IL-10 and D) IL-2. Values in the graphs represent the average pg/mL ± SD of PCLS from 4 different donor lungs normalized to a medium only control sample.
