Hemacolor staining kit

Cell migration was assessed in a 48-well microchemotaxis chamber (Neuroprobe, Gaithersburg, MD, USA) as described previously [19 (link)]. The chamber consisted of acrylic top and bottom plates, each containing 48 matched wells. Twenty-six microliters of FBS-free medium containing tested substance (10 μg/ml) were filled in wells of the bottom plate. Wells filled with medium containing 0.0001 % of acetic acid served as control. Subsequently, the bottom plate was covered with a polycarbonate filter with 8-μm pore size (Neuroprobe, Gaithersburg, MD, USA) and the top plate was applied so that each well corresponded to that of the bottom plate. 1 × 10⁴ cells resuspended in 50 μl FBS-free medium were added to each well of the top plate and the whole chamber was incubated at 37 °C in humidified air with 5 % CO₂ for 8 h. After incubation, cells on the upper surface of the filter were removed over the wiper blade and the filters were then fixed with methanol and stained using Hemacolor staining kit (Merck, Darmstadt, Germany). The cells migrated across the filter were counted under a light microscope at high-power magnification (×100) to measure transmigration in each well. Four fields were counted in each well and the total number was calculated. Four wells were used for each group; experiments were repeated in triplicate.

To analyze chemotaxis, a specific 96‐well cell migration system (Neuro Probe) was applied as previously described.^[54] Briefly, the chemotactic stimulus was added in a volume of 37.5 µL per well. A membrane filter with 5 µm pores was placed onto the plate. 3 × 10⁴ cells in 40 µL diet medium consisting of VLE‐RPMI (Biochrom) and 0.1% autologous serum were added on the membrane. Chemotaxis was induced by 50 or 100 µg mL⁻¹ NID1 or without stimulus (negative control). MCP‐1 (50 ng mL⁻¹, Miltenyi Biotec) served as positive control. After incubation for 3 h at 37 °C in a 5% CO₂ incubator, the membrane was carefully removed. Monocytes that adhered to the membrane were fixed with methanol (Merck) and labeled with Hemacolor staining kit (Merck). After microscopic documentation using ProgRes CapturePro 2.8.8 (Jenoptik), the number of migrated monocytes was determined using ImageJ Version 1.4.3.67 (National Institutes of Health). The number of migrated cells was normalized to the negative control.

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.