Reastain quick diff kit

To recover the airway cells and immune mediators, BAL was performed^{68 (link)}. One ml PBS supplemented with 0.5 mM EDTA was used to flush the lungs three times. The BAL was centrifuged, and supernatant stored at −80 °C. The cells were incubated 2 min in ACK buffer (0.15 M NH₄Cl, 1.0 mM KHCO₃, 0.1 mM Na₂EDTA) to lyse red blood cells (RBCs) and the cell number was determined by Trypan Blue (Thermo Fisher Scientific) exclusion of dead cells. Cells obtained from the BAL were termed airway cells throughout. Airway cells were either stained for flow cytometry or the cellular composition was determined by spinning 1–2 × 10⁵ cells onto a microscope slide (Thermo Scientific) at 450 rpm for 5 min using Cytospin 4 Cytocentrifuge (Thermo Fisher Scientific). Slides were H&E stained using Reastain Quick-Diff kit (Gentaur), according to the manufacturer’s instructions. Neutrophils were imaged using a light microscope and determined by nuclear morphology.

Mouse pulmonary fibroblasts (no. M3300-57, ScienCell) were cultured in poly-l-lysine-coated dishes and treated with 10 ng/ml transforming growth factor-β1 (TGF-β1; no. AF-100-21C, Peprotech) for 48 h to induce differentiation to myofibroblasts. Cells were transfected with either scrambled siRNA or two different siRNAs targeting Nrp1 (siRNA cat. nos. 70800 and s70802, respectively; Thermo Fisher Scientific) using RNAiMAX transfection reagent ( no. 13778075, Thermo Fisher Scientific). Transfected cells were seeded at a count of ~2.5 × 10⁴ cells/Transwell insert (8.0-μm pore size, no. 353097, Falcon) and left to migrate for 20–24 h in response to either serum-free media or serum-free media with 50 ng/ml PDGF-AA (100-13A, Peprotech). Nonmigrated cells were removed, and Transwell inserts were stained using the REASTAIN Quick-Diff Kit, according to manufacturer’s instructions (no. 102164, Gentaur), and migrated cells were counted under a Leica stereo microscope.

Matrigel invasion assays were carried out using BD Biocoat^TM growth factor reduced Matrigel® Invasion Chambers (PET 8.0 µm, Corning) according to manufacturer's instructions. Briefly, cells were serum-starved for 2 h, detached using cell dissociation buffer (GIBCO), and 3 × 10⁵ cells in serum free medium were added to the upper chambers. Chemoattractant (medium containing 10% (v/v) FBS) was added to the lower wells. The chambers were placed at 37°C in 5% CO₂ for 20 h. Non-invading cells in the upper chamber were scraped off and the migrated cells on the bottom of the chamber were fixed and stained using REASTAIN Quick-Diff kit (Gentaur Molecular Products) according to manufacturer's instructions.