Primary normal human bronchial epithelial (NHBE) cells were purchased from Lonza (Walkersville, MD, USA). We used NHBE cells from at least three different donors. NHBE cells were seeded in collagen-coated 24-well plates and were maintained in serum-free bronchial epithelial cell growth medium (BGEM, Lonza, Walkersville, MD, USA). Before stimulation, the NHBE cells were cultured in BEGM without hydrocortisone for 24 h. At confluency, submerged NHBE cells were stimulated with poly(I:C) (R&D Systems, Minneapolis, MN, USA), interferon (IFN)-γ (R&D Systems, Minneapolis, MN, USA), and interleukin (IL)-4 (R&D Systems, Minneapolis, MN, USA) for 24 h. Then, total RNA was isolated from the cells.
The Eol-1 human eosinophilic leukemic cell line was obtained from the Riken Cell Bank (Tsukuba, Japan). The cells were maintained in RPMI1640 medium (Gibco Laboratories, Grand Island, NY, USA) supplemented with 10% defined fetal bovine serum (Gibco) and 20 mM Hepes buffer (Gibco) in a humidified atmosphere with 5% CO2 at 37 °C as described previously [23 (link)]. For the cytokine stimulation study, 5 × 105 Eol-1 cells were incubated with a varying concentration of IL-5 (R&D Systems, Minneapolis, MN, USA) and IL-33 (R&D Systems, Minneapolis, MN, USA) for 24 h. Total RNA from the cultured Eol-1 cells was extracted, and single-strand cDNA was synthesized.
The Eol-1 human eosinophilic leukemic cell line was obtained from the Riken Cell Bank (Tsukuba, Japan). The cells were maintained in RPMI1640 medium (Gibco Laboratories, Grand Island, NY, USA) supplemented with 10% defined fetal bovine serum (Gibco) and 20 mM Hepes buffer (Gibco) in a humidified atmosphere with 5% CO2 at 37 °C as described previously [23 (link)]. For the cytokine stimulation study, 5 × 105 Eol-1 cells were incubated with a varying concentration of IL-5 (R&D Systems, Minneapolis, MN, USA) and IL-33 (R&D Systems, Minneapolis, MN, USA) for 24 h. Total RNA from the cultured Eol-1 cells was extracted, and single-strand cDNA was synthesized.