Diff quik stain kit

The collection of BAL fluid was performed 24 h after the last OVA challenge as described previously (Onodera et al., 2017 (link)). A total of 100,000 viable BAL cells were cytocentrifuged onto slides by a Cytospin 4 (Thermo Fisher Scientific) and stained with a Diff-Quik Stain Kit (Sysmex). 200 leukocytes were counted on each slide. The cell types were identified using morphological criteria.

As mice of the AR group have systemic allergies, pulmonary inflammation should also be induced. On the other hand, the pulmonary parenchyme of the LAR group should be normal, because mice in this group were induced local nasal inflammation without systemic atopy using only a small amount of OVA. To confirm that this experimental model was successfully made, we conducted a BAL fluid analysis.
In order to collect BAL fluid, the trachea was first cannulated using polyethylene tube, and then the lung was lavaged with 800 μL of Hank's balanced salt solution (HBSS; ThermoFisher Scientific) thrice. The fluid was centrifuged at 3,000 ×g for 15 minutes at 4°C, and the supernatant was stored immediately at −80°C for measuring the levels of cytokines. The resulting pellet was suspended immediately in saline for cell counting.
Total cell numbers were determined in duplicate using a hemocytometer. Subsequently, an aliquot of 100-200 μL of the BAL fluid was centrifuged in a Cytospin 2 cytocentrifuge (Shandon Scientific, Pittsburgh, PA, USA). The differential cell counts of eosinophils, neutrophils, and lymphocytes were determined from the centrifuged preparations that were stained using the Diff-Quik stain kit (Sysmex Corp., Kobe, Japan) by counting 500 or more cells from each sample at 200× magnification.

Cells were suspended in RPMI 1640 medium and placed in the upper compartment of an 8 μm Transwell^® (3.2 mm diameter; Neuro Probe, Gaithersburg, MD, USA). The lower compartment was filled with RPMI 1640 medium supplemented with FBS. After 24 h, the filter was washed with PBS and the migrated cells on the filter membrane were stained using a Diff-Quik Stain Kit (Sysmex, Tokyo, Japan). For invasion assays, the upper compartment of an 8 μm Transwell^® (6.5 mm diameter; Costar, Cambridge, MA, USA) was coated with Matrigel^® (1 mg/ml) before starting the assay. Each assay was conducted at least three times, and three random fields using 20 × magnification were analyzed for each filter membrane (21 (link)).

Transwell plate with 8 μm pore filters (Corning, USA) was used to evaluate the migratory ability of rMSCs. rMSCs (3 × 10⁴ cells) were seeded into the upper chamber with a mixture of SFM, NCM and HCM added to the lower chamber. Following incubation for 12 h and 24 h at 37°C in 5% CO₂, rMSCs that had not migrated through the upper side of filters were scraped off with a cotton wool swab. The filters were then stained with Diff-Quik stain kit (Sysmex, Japan), and the cells that had migrated to the lower side were counted using a light microscope (Nikon Co., Japan) at 100× magnification. The migration assay was conducted in triplicate.