Implication of NADPH Oxidases in the Early Inflammation Process Generated by Cystic Fibrosis Cells
Figure 2
Apoptosis of IB3-1defective CFTR cells as compared with S9 control cells. (a) Cells were plated on 96-well microplates and treated by the different isotonic, hypotonic, or hypertonic media, then incubated for 5βh at 37Β°C, and apoptosis was measured using yopro-1 probe as described in Section 2. Results were obtained in arbitrary fluorescence units (AFUs) and were expressed as the percentage of fluorescence signal obtained for each cell type in hypotonic and hypertonic conditions as compared with that obtained in isotonic conditions. AFUs of IB3-1 and S9 cells incubated in isotonic conditions were 25664 Β± 807 and 27857 Β± 980, respectively. t-butyl hydroperoxide (-BHP, 2βmM) was used as an oxidative stress inducer. Results are mean Β± SEM (). significantly different from S9 (); *significantly different from IB3-1 or S9 in isotonic medium, respectively (). (b) Microscopic examination of IB3-1 and S9 after exposure to hypertonic medium for 5βh, then loaded with 10βM Yopro-1. Cells were examined in fluorescence microscopy (inverted microscope Olympus IMT2) (magnification 300). (c) The mitochondrial apoptotic pathway was detected as a reduction of DiOC6 incorporation in mitochondrial membranes after incubation of IB3-1 cells and S9 cells in in the various ionic conditions for 5 hours, in the presence or absence of 2.5βM diphenyleneiodonium (DPI), as described in Section 2. t-butyl hydroperoxide (t-BHP, 2βmM), used as a control of oxidative stress, was incubated for 1 hour with both cell lines. Then cells were loaded with 40βM DiOC6 and further incubated for additional 15βmin at 37Β°C. Cells were analysed by flow cytometry, and retention of DIOC6 was expressed in percentage of DiOC6low cells. Values are means SEM (). significantly different from S9 in the same conditions (); *significantly different from IB3-1 or S9 in isotonic medium, respectively (); #significantly different from non treated with DPI in the same medium ( ).
