Cells were incubated for 1 h at 37°C with 125 µM substrate. The fluorescence of the cleaved reporter group was measured at
an excitation wavelength of 360 nm and an emission wavelength of 465 nm. Camptothecin (an extract of Target Selective Inhibitor Library mw the Chinese tree Camptotheca acuminata) is a potent inhibitor of topoisomerase I, a molecule required for DNA synthesis. Camptothecin has been shown to induce apoptosis and was therefore used for positive controls. The percentage of apoptotic and nectrotic cells was quantified by performing a cell staining with annexin V and 7-amino-actinomycin D (7-AAD) (PE Annexin V Apoptosis Detection Kit I; BD Biosciences, Franklin Lakes, NJ, USA). Apoptosis was quantified directly with annexin V, measuring the translocation of phospholipids phosphatidylserines from the inner to the outer leaflet of the plasma membrane in apoptotic cells. The loss of membrane integrity in late apoptotic or necrotic PLX4032 chemical structure cells was assessed by 7-AAD staining. 7-AAD intercalates into double-stranded nucleic acids. It is excluded by viable cells, but can penetrate cell membranes of dying or dead cells. For
analysis, a fluorescence activated cell sorter (FACSCalibur) flow cytometer (Becton Dickinson) was used. Results are expressed as median and the error bars are plotted as median with interquartile range for the caspase assays. Values from stimulated cells are shown as percentage compared to control values of 100%. All experiments were conducted at least four times. Analysis of variance (anova) and Kruskal–Wallis multiple comparison tests were performed to assess the statistical significance of differences, using GraphPad Prism version 4·0 software (San Diego, CA, USA). For flow cytometry analysis, box-plots were designed using the spss program. P-values <0·05 were considered significant. To determine a possible effect of hypoxia on apoptosis in
alveolar macrophages and neutrophils caspase-3 as the key enzyme in the final pathway was determined as well as caspase-8 and -9 to distinguish between intrinsic and extrinsic pathways. Interestingly, the two cell types, although belonging to the group of effector cells, did not experience the same changes. While the Carnitine palmitoyltransferase II apoptosis rate did not change under hypoxic conditions in alveolar macrophages at early time-points compared to control cells, caspase-3 activity increased by 80% in the LPS group and caspase-8 activity showed a threefold increase in the same group after 4 h (P < 0·05) (Fig. 1a). After 8 h, caspase-3 activity was enhanced by 240%, caspase-8 activity by 148% and caspase-9 activity by 85% in the LPS group (P < 0·05) (Fig. 1b). Figure 1c shows the 24 h caspase-3, -8 and -9 activities with no significant changes, except again for the LPS group, where caspase-3 level was increased by 277%, caspase-8 level by 41% and caspase-9 by 198% (P < 0·05).