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. 2015 Dec 14;212(13):2289–2304. doi: 10.1084/jem.20150388

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© 2015 Phong et al.

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Figure 1. — Tim-3 antibodies enhance IgE/Ag-mediated IL-6 and TNF production in BMMCs with a modest effect on degranulation. (A–C) BM mast cells (BMMCs) were generated from C57BL/6 mice, sensitized with IgE overnight, and then stimulated with either Ag alone, Ag with Tim-3 pAb, mAbs 5D12, 3E3, or the respective isotype controls, for 6 h. Culture supernatants were collected and analyzed for IL-6 (left) or TNF (right) by ELISA. Results shown are representative of three independent experiments performed in duplicates. (D) BMMCs were sensitized overnight with IgE, stimulated with DNP₃₂-HSA alone or together with isotype control, or Tim-3 pAb, for 30 min. Degranulation was assessed by measurement of β-hexosaminidase release (left); alternatively, cells were labeled with LysoTracker Deep Red, sensitized with IgE for 1 h, and stimulated with Ag in the presence of isotype control or indicated amount of Tim-3 pAb for 90 min before Annexin V staining and flow cytometry analysis (right). Results shown are average of three independent experiments performed in triplicates. (E) BMMCs were sensitized and stimulated for 6 h with Ag in the presence of isotype control, Tim-3 pAb (left), or 5D12 (right), with or without Fc block (2.4G2). Supernatants were harvested and analyzed by ELISA for IL-6. Results shown are based on duplicate samples and are representative of two independent experiments. *, P < 0.05; **, P < 0.005; ***, P < 0.0005.