Diff quik staining

Twenty-four hours after the final OVA challenge, blood specimens were collected from retro- orbital plexus, and one drop was smeared on a slide and stained with Diff-Quik staining (Sysmex Co., Kobe, Japan). Serum was obtained by centrifugation at 1000 g for 10 min at 4 °C (Centrifuge 5403; Eppendorf, Hamburg, Germany), and then serum was stored immediately at −80 °C for future analysis. The serum levels of OVA-specific IgE, OVA-specific IgG_1, and OVA-specific IgG₂ were measured using enzyme-linked immunosorbent assay (ELISA), and the optical density was measured at 450 nm in accordance with the protocol provided by the manufacturer.

For transwell migration assays, NPDFs (1.5 × 10⁴) were seeded onto Transwell chambers (Corning Life Sciences, MA) and cultured for 48 hours in DMEM containing 10% FBS, TGF-β1 (5 ng/mL), and/or 1,25(OH)₂D₃ (100 nM). Cells on the upper surface of the membrane were removed using cotton swabs, and then the cells on the lower surface of the membrane were stained using Diff-Quik staining (Sysmex, Kobe, Japan). Images of stained cells from five selected views were captured under microscopy at 200x magnification (Olympus BX51; Olympus, Tokyo, Japan).

For migration assay using RAW264.7 cells, 10⁵ cells were seeded in cell culture inserts with 8 μm pores, and 0, 10 and 50 µg/ml of recombinant VCAM-1 was added into the media in the bottom well. After incubating 10 h with or without anti-VCAM-1 neutralizing antibody (Millipore, Burlington, MA), migrated cells were stained by Diff-Quik staining (Sysmex, Kobe, Japan).
For coculture of RAW264.7 cells and PDAC cells, 1 × 10⁴ RAW264.7 cells were seeded in cell culture inserts with 0.4 μm pores and 5 × 10⁴ PDAC cells were seeded in the bottom wells. After incubating with gemcitabine for 24 h, CCK-8 (Dojindo, Kumamoto, Japan) was used to measure cell viability.

Blood was collected from the abdominal vein of anesthetized mice using Alfaxan (Jurox Pty Ltd.), and the levels of ovalbumin-specific IgE (500840, Cayman Chemical, Ann Arbor, MI, USA), histamine (ENZ-KIT140-0001, Enzo Life Science, Farmingdale, NY, USA), and IL-13 (DY413, R&D Systems, Abingdon, UK) in serum were measured using commercial enzyme-linked immunosorbent assay (ELISA) kits following manufacturers’ instructions. NALF was collected by washing the nasal cavity with 10 mL of PBS after creating an incision in the bronchial tubes. The total number of cells in the NALF was measured using a cell counter (Thermo Fisher Scientific, Waltham, MA, USA). After centrifugation using Cytospin (Hanil, Seoul, Korea), Diff-Quik staining (Sysmex, Chuo-ku, Kobe, Japan) was performed according to the manufacturer’s instructions, and the numbers of eosinophils in each group were measured and compared using a light microscope (Olympus, Tokyo, Japan).

After physiological analysis, 1 mL of sterile saline was injected through the needle using a syringe, the lungs were washed, and the lavage fluid was retrieved three times to obtain bronchoalveolar lavage fluid (BALF). The collected BALF was centrifuged at 450 × g for 10 min at 4℃. The pellet was dissolved in 1 mL of PBS after separating the supernatant, and cells were counted by a LUNA cell counter (Logos Biosystems, Korea). Diff-Quik staining (Sysmex, Japan) was used to measure leukocyte fractions of cells in BALF.

The properties of cell migration and invasion were performed using a 24-well hanging insert (SPL, #35224) for transwell migration and invasion assay^{46 (link)}. Before seeding the cells in a hanging well, cells were pretreated with siRNA or overexpression with Kv3.4 or TGF-β for the treatment group, and control siRNA or control vector or 2 mg/ml BSA for the control group. After cell detachment, the appropriate numbers of cells were plated on the upper side of a cell-permeable membrane with FBS-free media, and media containing 10% FBS were placed on the lower chamber of the membrane. Following incubation of 24 h for migration, the migrated cells were stained using Diff-Quik staining (Sysmex Corporation, Hyogo, Japan, #38721) and imaged by microscope (CKX53, Olympus). For invasion assay, 120 μl of 1.0 mg/ml diluted Matrigel (Corning^®, Arizona, USA, #356234) was coated on the upper insert membrane for 1 h at 37 °C before cell plating. After plating the appropriate numbers of cells for 48 h, invaded cells were stained using Diff-Quik staining, and imaged. Migrated and invaded cells were randomly photographed in five fields per each membrane and quantified with Fiji ImageJ software (National Institutes of Health).