Microscopy hemacolor

Migration and invasion assays were performed as described previously [17 (link)]. Collagen (3 μg/μL; Sigma-Aldrich, St. Louis, MO)-coated transwell plates (8 μm pore; Corning Inc., Corning, NY, USA) were used for detection of cell migration and matrigel-coated transwells (1 mg/mL; Invitrogen Life Technologies, Carlsbad, CA, USA) were used for detection of cell invasion. IR-treated or non-treated A549 cells (2 × 10⁴) in 200 μL serum-free medium were added to 0.1% bovine serum albumin and seeded onto the upper chamber. The lower chamber was filled with 1 mL of 20% fetal bovine serum (FBS) medium. After incubation for 24 h with 5% CO₂ at 37 °C, cell staining was performed with Microscopy Hemacolor (Merck, Whitehouse Station, NJ, USA), according to the manufacturers’ protocols. Stained cells were counted under a microscope and statistical analyses were performed.

Cells were cytospun (Cytospin 3, 300 rpm for 5 minutes; Shandon, Runcorn, UK), air-dried, stained with Microscopy Hemacolor (Merck, Millipore, Billerica, Massachusetts, USA), visualized on a Leica DMLB microscope with DFC490 camera (Leica, Wetzlar, Germany) and processed using QWin Software (Leica).

BALFs were centrifuged (200g for 5 min at 4 °C) to pellet cells and erythrocytes were lysed by hypotonic shock. Cells were then resuspended in 500 µL ice-cold saline-EDTA and total cell counts were determined using a Neubauer’s chamber. Differential cell counts were assessed on cytologic preparations obtained by cytocentrifugation (Cytospin 4, Thermo Scientific) of 200 µL of diluted BALFs (250,000 cells/mL in ice-cold saline-EDTA). Slides were stained with Microscopy Hemacolor® (Merck) and at least 400 cells were counted for each preparation. Eosinophil, neutrophil, lymphocyte and macrophage numbers were then expressed as absolute numbers from total cell counts.