Poly l lysine coated cover glasses

TG cells were isolated from neonatal Wistar rats (7 days old) (Kawaguchi et al., 2015 (link)) that were under pentobarbital sodium anesthesia (50 mg/kg) following the administration of isoflurane (3.0 Vol%). TG cells were dissociated by enzymatic treatment with Hank’s balanced salt solution (Life Technologies, Grand Island, NY, USA) containing 20 U/mL papain (Worthington Biochemical Corporation, Lakewood, NJ, USA) for 20 min at 37°C, which was followed by dissociation by trituration. After dissociation, the TG cells were plated on 35 mm-diameter dishes (Corning Incorporated Life Sciences, Tewksbury, MA, USA) and cultured for 48 h at 37°C (95% air and 5% CO₂). The primary cells were cultured in Leibovitz’s L-15 medium (Life Technologies) containing 10% fetal bovine serum, 1% penicillin-streptomycin (Life Technologies), 1% fungizone (Life Technologies), 26 mM NaHCO₃, and 30 mM glucose (pH 7.4). For the immunocytochemistry, TG cells were subjected to primary culture on poly-L-lysine-coated cover glasses (Matsunami Glass Ind., Ltd., Osaka, Japan).

Human Nasal Epithelial Cells (HNEpCs) (Promocell, Rockville, MD, USA) were cultured on poly-L-lysine coated cover glasses (Matsunami, Osaka, Japan) in Airway Epithelial Cell Growth Medium (Promocell) and maintained in 5% CO₂ and 95% air at 37 °C. Twelve hours after stimulation with TDI-HSA, HNEpCs were fixed with 4% sucrose-containing 4% paraformaldehyde (Sigma Chemical Co.) for 20 min. The fixed cells were permeabilized with 0.2% Triton X-100/PBS (Sigma Chemical Co.) for 5 min and blocked with 10% goat serum/PBS for 1 h. Then, an anti-IL-33 antibody (Nessy-1, 1:250, Enzo Life Sciences, Farmingdale, NY, USA) was applied overnight at 4 °C. IL-33 was visualized by isotype-specific secondary antibody conjugated with Alexa 488 (1:200, Molecular Probes, Eugene, OR, USA). A fluorescent microscope (Axio Observer, Carl Zeiss, Oberkochen, Germany) was used to obtain fluorescent images.