Shp099

Total, naïve or memory peripheral blood B cells were stimulated with the F(ab)₂ fragment of an affinity purified mouse anti-human μ heavy chain antibody [F(ab)₂ anti-IgM, 1μg/ml] followed by soluble CD40 ligand (CD40L, 2μg/ml), in the presence of a mouse anti-human SLAMF1 mAb (5μg/ml) or a mouse IgG1κ isotype control (5μg/ml) for the indicated time points. In some experiments, cells were cultured in the presence of a pharmacological inhibitor against SHP-2 (SHP099, purchased from Cayman Chemical).
For cytokine detection, cells were re-stimulated with PMA (25ng/ml) and ionomycin (0.5μg/ml) in the presence of Brefeldin A (GolgiPlug 1μg/ml) for the final 6h of culture. For B cell differentiation, naïve B cells were stimulated as mentioned above in the presence of IL-4 (10ng/ml, Peprotech) for 7d. IL-4 was replenished every 3d. For immunoglobulin production, naïve B cells (50×10³/200μl, 96-U bottom, complete medium) were stimulated with F(ab)₂ anti-IgM (1μg/ml), CD40L (2μg/ml) and IL-4 (10ng/ml), in the presence or absence of SLAMF1 mAb (5μg/ml) or an isotype control, for 12 days.

Tyrode solution contained (in mM): NaCl, 140; KCl, 5; CaCl₂, 1; MgCl₂, 1; NaHCO₃, 5; HEPES, 10; Glucose, 10; sodium pyruvate, 10; pH adjusted to 7.4 with NaOH. Sucrose (500mM) was used as a sweet tastant. Recombinant murine leptin (20 ng/ml; PeproTech, Cat# 450-31), wortmannin (10 nM; Tocris Bioscience, Cat# 1,232), LY294002 (30 μM; Sigma-Aldrich, Cat# L9908), SHP099 (10 μM; Cayman Chemical, Cat# 20,000), and stattic (10 μM; Axon Medchem, Cat# 2,314) were applied from the basolateral side of TCs. Other chemicals were purchased from Wako Pure Chemical Industries or Nakarai Tesque. These concentrations of agents were selected as used in previous studies (Chen et al., 2016 (link); Mirshamsi et al., 2004 (link); Schust et al., 2006 (link); Yoshida et al., 2015 (link)).