GlcNAc increases MC susceptibility to IgE-mediated stimulation. (A) Degranulation of bone BMMC KITD814V after treatment with or without 100 mM GlcNAc was measured by assessment of β-hexosaminidase release (n = 3). Cells not stimulated with DNP-OVA or ionomycin were considered negative controls, whereas ionomycin stimulation was used as a positive control for MC degranulation ability. (B) Degranulation of MCs was measured by annexin V staining upon stimulation with IgE and antigen complexes for 30 minutes. (C) Intracellular staining (left) or release (right) of TNF-α in MCs stimulated with IgE and antigen incubated with PBS or GlcNAc. The results of 1 representative experiment are shown. SSC, side scatter. (D) Same as panel C for intracellular IL-13. (E) Same as panel C for intracellular IL-6. (F) BMMC KITD814V were transfected with a validated small interfering RNA designed against the OGlcNAcase (OGA) enzyme for 48 hours and degranulation was assessed as described in panel A. (G) Schematic representation of the experimental design for passive cutaneous anaphylaxis. (H) Acute dye extravasation was measured by assessment of the optical density (OD) at 650 nm (n = 20 in each group). The values in the graphs are presented as the means ± SD (nonsignificant, *P < .05, **P < .01, ***P < .001; 2-tailed, unpaired Student t test).