Effect of Tumor Necrosis Factor Family Member LIGHT (TNFSF14) on the Activation of Basophils and Eosinophils Interacting with Bronchial Epithelial Cells
Adhesion of basophils or eosinophils onto BEAS-2B cells. Coculture of BEAS-2B cells (1 × 105 cells) and basophils/eosinophils (3 × 105 cells) using transwell inserts or BEAS-2B cells alone were stimulated by LIGHT (0–100 ng/mL) for 24 h. Basophils/eosinophils were removed and freshly isolated basophils/eosinophils (3 × 105 cells) were added into the corresponding well containing the adherent BEAS-2B cells (1 × 105 cells) for 1 h incubation. The ratio of (a) basophils or (b) eosinophils adherent onto BEAS-2B was analysed using flow cytometry as described in Section 2. Results are expressed as the mean plus SEM of three independent experiments with three blood samples. *, ***. BEAS-2B: BEAS-2B cells alone were treated with LIGHT prior to adhesion analysis; Co-BAS + BEAS-2B: Coculture of BEAS-2B cells and basophils were treated with LIGHT before BEAS-2B cells were used for adhesion analysis; Co-Eosi + BEAS-2B: Coculture of BEAS-2B cells and eosinophils were treated with LIGHT before BEAS-2B cells were used for adhesion analysis. In the above adhesion assay, basophils/eosinophils and BEAS-2B cells were analyzed separately using flow cytometry. (c) Representative histograms of the flow cytometric analysis of the number of adherent basophils in coculture gated by the specific cell surface basophilic marker CD203c were shown. (d) Representative dot plots of the flow cytometric analysis of the number of adherent eosinophils in coculture gated by the SSC and FSC were shown.
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