Dy3548b

All quantification methods were detailed in our previous study^{21 (link)}. Briefly, the QPLEX^TM kit utilized Quantamatrix’s multiplex diagnostics platform (QMAP; Quantamatrix Inc., Seoul, Republic of Korea)²⁹ . First, human plasma samples (singular) were diluted in diluent buffer and incubated with the coded beads and biotin-conjugated detection antibodies (angiotensin-converting enzyme, ACE, DY929, R&D Systems, Minneapolis, USA; galectin-3 binding protein, LGALS3BP, DY2226, R&D Systems; periostin, POSTN, DY3548B, R&D Systems; Aβ1−40, 014-26923, Wako, Japan). The immunocomplexes were washed twice with washing buffer at a Biotek-510 magnetic wash station (Biotek, VT, USA). Diluted R-phycoerythrin-conjugated streptavidin was added to each well. After three washes, the immunocomplexes were resuspended in 100 μl of washing buffer by tapping and automatically analyzed using the QMAP^TM system. The intra/interassay coefficients of variation and limits of detection were as follows: ACE, intra: 4.9%, inter: 5.1%, 0.22 ng/ml; LGALS3BP, intra: 1.1%, inter: 5.7%, 0.04 ng/ml; POSTN, intra: 9.0%, inter: 6.0%, 0.034 ng/ml; and Aβ1−40, intra: 5.3%, inter: 3.9%, 0.50 pM.

Epithelial cell function was assessed by the production of typical cytokines of type 2 inflammation, representatively IL-33 and periostin. Concentrations of IL33 and periostin in culture supernatants were measured by ELISA according to the manufacturer’s instructions (Invitrogen, cat #: BMS2048TEN, Carlsbad, CA, USA; R&D Systems, cat #: DY3548B, Minneapolis, MN, USA) and as described previously [21 (link)]. To obtain the supernatants, 5,000 cells were seeded in a 24-well plate with 1 ml BEGM, and medium was renewed every 2 days until 70%–80% confluence. Then 1 ml of BEGM was added, and supernatants were collected after 24, 48, and 72 h. Cells from 2nd or 3rd passage were used for the analysis.

Epithelial cell function was assessed by the production of typical cytokines of type 2 inflammation, representatively TSLP and periostin. Concentrations of TSLP and periostin in culture supernatants were measured by ELISA according to the manufacturer’s instructions (DY1398-05 and DY3548B R&D Systems, MN, USA) and as described previously [15 (link)]. To obtain the supernatants, 5,000 cells were seeded in a 24-well plate with 1 mL BEGM, and the medium was renewed every 2 days until 70–80% confluence. Next, 1 mL of BEGM was added, and supernatants were collected after 24, 48, and 72 h. Cells from the 2nd or 3rd passage of cryopreserved Polyps 3–5 were used for the analysis.