Fluoprobes 488

Neutrophil elastase was detected using anti-human NE antisera raised in rabbits [24 (link)]. Approximately 3 x 10⁵ cells were seeded onto Superfrost slides and treated as described previously [24 (link)]. Briefly, primary rabbit anti-peptide antibodies specific for each human protease were diluted 1/200 (anti-NE) and bound antibodies were detected with an anti-IgG coupled to FluoProbes^®-488 (Interchim, Montluçon, France). DRAQ5™ (Interchim, Montluçon, France) (10 μM) was used to detect dsDNA. Samples were examined with an Olympus FV 500 confocal microscope (Olympus, Rungis, France). Purified peripheral blood neutrophils were activated by incubation with P. aeruginosa at a MOI (multiplicity of infection) of 1:20 for 1 h at room temperature before evaluation as described in Brea et al [24 (link)].

After exposure of HAoECs to one cycle of IH, cells were incubated (1 hour, room temperature) in blocking buffer (1% non-fat dry milk in physiological buffer solution). After 3 washes, cells were incubated overnight (4 °C) with anti-IL-6 and anti-VAMP (1:100, Santa Cruz Biotechnology) antibodies. Three washes were followed by incubation (2 hours, room temperature) with secondary mouse fluorescence-labelled antibody Fluoprobes FP 546 and secondary rabbit fluorescence-labelled antibody Fluoprobes 488 (1:500, Interchim, San Diego, CA). Images were then taken using confocal microscope and analyzed with Zeiss software.