Diff quick stain

Mice were anesthetized by i.p. injections of sodium pentobarbital (70 mg/kg), their tracheas were cannulated, and BALF was collected by instilling 1 ml of sterile PBS into the trachea twice via a blunted 18-gauge needle. Recovery of the fluid was consistently 70–80% of the total. For determination of total number of cells in BALF, the BALF was then centrifuged at 1,000 rpm for 10 min at 4°C, and the cell-free supernatant was collected and stored at -80°C until analysis. The cell pellets were resuspended in 1 ml PBS and counted with an automated cell counter (TC50 Automated Cell Counter) (Bio-Rad, Hercules, CA). The remaining cells were centrifuged and resuspended in a small amount of a buffer at ~10⁶ cells/μL. Five μL of the cell suspension was spotted on slide glasses, and extended with a cover glass to make a thin film of the cell suspension. Slide glasses were air-dried and stained with Diff-Quick stain (Sysmex, Kobe, Japan). The percentages of macrophages, lymphocytes and neutrophils were determined by counting a minimum of 200 cells per slide under a BX 41 bright-field microscope. Soluble collagen content in BALF was determined using the Sircol Soluble Collagen Assay kit (Biocolor, Northern Ireland, UK) according to the manufacturer’s instructions. Total protein concentrations in BALF were determined by Lowry’s method as described previously [25 (link)].

Cell growth was assessed by counting the number of cells with a hematocytometer or by the Cell Titer-Glo assay (Promega, Madison, WI, USA). Cell migration was examined using 8 μm pore size polycarbonate transwell filters (Becton Dickinson). After serum starvation for 48 h, the cells were seeded in serum-free media on the upper side of a transwell chamber and allowed to migrate towards media containing 10% FBS in the lower chamber for 24 h. After migration, the cells on the lower side of the membrane were fixed, stained with Diff-Quick stain (Sysmex, Kobe, Japan), and counted. The migration indices were calculated as the mean number of cells in 5 random fields at 20× magnification.

The matrigel invasion assay was performed using the Biocoat Matrigel Invasion Chamber from BD (Franklin Lakes, NJ, USA) according to the manufacturer protocol. In brief, 5 × 10⁴ cells were plated in the upper chamber in serum-free media. The bottom chamber contained DMEM media with 10% FBS. After 24 h, the bottom of the chamber insert was fixed and stained with Diff-Quick stain (Sysmex, Kobe, Japan). Cells on the stained membrane were counted under a microscope. Each membrane was divided into four quadrants and an average from all four quadrants was calculated. At least two chambers were used for each group. Each matrigel invasion assay was performed at least three times. Relative cell invasion was normalized to that of control cells.

Migration was analyzed by Boyden chamber assay using Transwell cell culture inserts (8.0 μm pore size; Costar, Cambridge, MA). Adjusted viable cell concentration was counted with trypan blue exclusion. Mouse embryonic fibroblasts were plated (5 × 10⁵ cells/ml in serum-free DMEM) in the upper chamber. The lower chamber containing various growth factors indicated in the figure legend. After 5 h, the cells remaining on the upper surface of the membrane were wiped off, and cells migrating to the lower surface in triplicate wells were visualized with Diff-Quick stain (Sysmex, Japan) and counted in each of four randomly chosen light microscopic fields at × 20 objective.

Nasal fibroblasts were seeded into transwell inserts with 8.0 μm pores (Sigma-Aldrich) on a 24-well plate. Serum-free DMEM was added to the bottom chamber. After 48 h, non-invasive cells were removed from the upper chamber, and invasive cells were stained with Diff-Quik stain (Sysmex). Then, migrated cells on the lower wall surface were fixed with methanol and stained with Diff-Quick stain (Sysmex) for 10 min. The number of cells invading the membrane was counted from 5 randomly selected visual fields using an inverted microscope (Olympus BX51; Olympus) at ×200 magnification.