Processed sputum was centrifuged at 37 × g for 3 min the supernatant was centrifuged at 253 × g for 10 min and then recentrifuged at 253 × g for 20 min to remove the cells and large debris, respectively. Two hundred μl of each MP-containing supernatant were frozen and stored at-80°C until characterization by flow cytometry and microbiological tests.