Kwik diff stain

Cells were serum starved overnight (0.1% DMEM), then 2 × 10⁵ cells were seeded on top of 8 μm transwell inserts (BD Biosciences, Mississauga, ON, Canada) with 0.1% DMEM and pre-coated with 1 mg ml⁻¹ Matrigel (Becton, Dickinson and Company, Mississauga, ON, Canada); 10% DMEM was used as a chemoattractant. After 24 h, cells that had invaded through the Matrigel-coated transwell inserts were fixed, stained by Kwik-Diff Stain (Thermo Fisher Scientific, Mississauga, ON, Canada) and number of invading cells counted under × 10 using a Leica DM LB2 microscope (Leica Microsystems, Richmond Hill, Ontario, Canada).

Sputum from patients with CF was obtained during a physiotherapy session while sputum from healthy subjects was induced by using the method of Pizzichini and colleagues, with minor modifications [26] (link). Sputum induction was achieved with an aerosol of hypertonic saline generated by the NE-U17 ultrasonic nebulizer (Omron). Healthy subjects equipped with a nose clip, inhaled increasing concentrations of saline (3, 4, and 5%) for 7 min each through a mouthpiece. We then asked subjects to rinse their mouth or swallow to minimise contamination with saliva. Finally, we invited them to cough sputum into a sterile container. Collected sputum was diluted 1:10 (w:v) in phosphate-buffered saline with 10 U/mL DNase (Pulmozyme 2500 U/2.5 mL – Roche, USA) and incubated under agitation, during 30 min, at 37°C. Then, it was filtered with 48 µm pore nylon filter (Prosep, Belgium) and centrifuged at 450 g, during 5 min. Neutrophil elastase activity was measured in supernatant. Remaining supernatant was treated with PMSF Protease Inhibitor 2% v:v in order to inactivate elastase and was stored at -80°C. Cells were processed for cytospin (fixed in methanol and staining by Diff-Quick method (Kwik-Diff™ Stains, Thermo Fisher Scientific) and epithelial contamination was assessed. The blood was centrifuged at 650 g for 20 min, the serum was collected, and stored at -80°C.