Cytospin 3

Vehicle, azithromycin, and compound 4 were administered intraperitoneally at a dose of 200 mg/kg (b.w.) to 10-week-old male BALB/cJ mice (Charles River, Lyon, France) 2 h before intranasal challenge with 2 μg of LPS from E. coli. Then, 24 h after LPS application animals were euthanized and bronchoalveolar lavage was performed. Total number of cells in BALF was counted with a hematological analyzer (Sysmex SF 3000, Sysmex Corp., Kobe, Japan). Percentages of neutrophils were determined by morphological examination of at least 200 cells on smears prepared by cytocentrifugation (Cytospin-3, Thermo Fisher Scientific Inc, Waltham, MA, USA) and stained with Kwik-Diff staining set (Thermo Fisher Scientific Inc., USA).

To collect bronchoalveolar lavage fluid (BALF), lung lavage was performed, using 1 mL of Hank’s balanced salt solution (HBSS) through a tracheal tube. The recovered BALF was centrifuged and resuspended in 300 µL HBSS. Total cell numbers were determined using a hemocytometer and trypan blue staining. BALF cells were centrifuged by cytocentrifugation (Cytospin 3; ThermoFisher Scientific, Waltham, MA, USA) and were pelleted to cytospin slides. The slides were stained with hematoxylin and eosin (H&E Hemacolor^®, Merck, Darmstadt, Germany), and a differential count of inflammatory cells was performed (200 cells per slide).

To collect bronchoalveolar lavage (BAL) fluid, the lungs were lavaged with 1 mL Hank’s balanced salt solution via the tracheostomy tube. BAL cells were counted with a hemocytometer, smeared by cytocentrifugation (Cytospin3, Thermo, Billerica, MA) at 1000 rpm for 3 min, and then stained with a Hemacolor Staining Kit (Merck, Darmstadt, Germany). BAL cells from each group were counted and classified as macrophages, lymphocytes, neutrophils, or eosinophils. To minimize the effects of subjective bias in the classification of the BAL cells, blind outcome assessment was used.
For protein extraction, lung tissues were homogenized in 20 mL/g tissue protein extraction reagent (Thermo Fisher Scientific Inc., Rockford, IL) using a tissue homogenizer (Biospec Products, Bartlesville, OK). Homogenates were incubated at 4°C for 30 min and then centrifuged at 1000 × g for 10 min. Supernatants were collected, passed through a 0.45-micron filter (Gelman Sciences, Ann Arbor, MI), and then stored at -70°C for assessment of cytokine levels. The measured cytokine levels were normalized to lung tissue weight and expressed as ng per mL per lung tissue.

One hundred microlitres of whole blood was removed at 2, 4 and 6 h post-infection and mixed with 1.5 mL 1X FACS Lysing Solution (BD Biosciences, Wokingham, UK) before incubating at room temperature for 20 min (n = 5). Tubes were centrifuged, the supernatant discarded, and the pellet resuspended in 100 µL PBS (Thermo Fisher Scientific, Loughborough, UK). Then, 80 µL of the suspension was placed into a cytospin funnel cartridge attached to a microscope slide and centrifuged in a Cytospin 3 (ThermoShandon, Thermo Fisher Scientific, Loughborough, UK) at 300 g for 3 min. Slides were stained with Red & Purple Microscopy Hemacolor (Merck, London, UK) before being mounted with coverslips. Slides were left to dry between each described step and were viewed using a Nikon eclipse 50i microscope at x1000 magnification under oil immersion (x100 objective). Neutrophils were identified, and the number of phagocytosed and non-phagocytosed neutrophils was counted in random fields of view as previously described [52 (link)]. The percentage of phagocytosis was recorded as the degree of phagocytosis.

Nasal lavage (NAL) was performed with 5 mL warm PBS (22–25 °C). A sample of at least 3 mL nasal lavage secretion was collected and filled into a precooled, sterile container. Before centrifugation (3800 rpm, 15 min, room temperature), samples were vigorously shaken. The pellet was suspended in PBS (phosphate bufferd saline). For each participant two cell smears were obtained by low-speed centrifugation (400 rpm, 6 min, room temperature) with a Cytospin 3 (Thermo Fischer, Waltham, Massachusetts, USA). The smears were stained with Giemsa stain (Sigma Giemsa Stain, Sigma-Aldrich Handels GmbH, Vienna, Austria) and mounted with Entellan® (Sigma-Aldrich Handels GmbH, Vienna, Austria). Cells were counted under light microscopy at 100× magnification. Cell counts were performed on both smears, with a total count of at least 200 cells per smear.
Mucociliary clearance time was measured via the saccharin test described by Andersen et.al. [40 (link)]. A saccharin particle was placed in the inferior turbinate of one nasal cavity at least 7 mm behind the anterior end of the turbinate. The participants were instructed to sit down and not to sniff, sneeze, cough, eat or drink.

Peritoneal lavage cells were washed at 500 ×g and resuspended in 2 mL RPMI. Total counts were carried out in a hemocytometer after a 1 : 10 dilution in Turk's solution. Differential counts were done on Giemsa-stained (ice-cold methanol-fixed, air-dried, and Giemsa-stained for 5 minutes) cytocentrifuge smears (500 rpm, 8 minutes in a Cytospin 3, Thermo Scientific, Waltham, MA), by counting at least 300 cells in 1000x magnification under oil.