Igedansyl clone 27 74

N-Fmoc-amido-dPEG₆-acid [Fmoc is also known as fluoren-9-ylmethoxycarbony] was purchased from Quanta BioDesign. N-Fmoc-Glu(OtBu)-OH, Boc-Lys(Fmoc)-OH, Fmoc-lys(ivDde)-OH, NovaPEG Rink Amide resin, HBTU [2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate], Fmoc-Arg(pfb)-OH and BSA (Bovine Serum Albumin) was purchased from EMD Biosciences. IgE^DNP (clone SPE-7), dansyl chloride, 1-Fluoro-2,4-dinitrobenzene (DNFB), DIEA (N,N-diisopropylethylamine), TFA (trifluoroacetic acid), Triisopropylsilane (TIS), hydrazine, Cholesterol, Dichloromethane, 2-proponol, ACN(acetonitrile) and piperidine were from Sigma and DMF (dimethylformamide) (>99.8%), chloroform, DiD fluorescent dye (3H-Indolium, 2-(5-(1,3-dihydro-3,3-dimethyl-1-octadecyl-2H-indol-2-ylidene)-1,3-pentadienyl)-3,3-dimethyl-1-octadecyl-, perchlorate), Minimum Essential Media was purchased from Thermo Fisher. IgE^dansyl (clone 27–74) were purchased from BD Biosciences. DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DSPE-mPEG2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt)), membranes and all mini extruder components were purchased from Avanti Polar Lipids (Alabaster, Al, USA). DNP-BSA conjugate was purchased from Invitrogen.

RBL cells and IgE^DNP were kindly provided by Dr. Wilson (University of New Mexico) and IgE^dansyl (clone 27–74) was purchased from BD Biosciences. RBL cells were maintained as described previously (11 (link)). For the degranulation assays, 100 μL of cells were plated at 0.5 x 10⁶ cells/mL in a 96 well plate, and were incubated for 24 hours followed by a two hour incubation with the indicated IgE antibody. Cells were washed immediately before experiments and were stimulated with the indicated concentrations of allergen for 60 minutes. When testing the allergy inhibitors, the inhibitor was added to cells 30 minutes prior to the allergen. Degranulation was detected spectroscopically by measuring the activity of the granule stored enzyme β-hexosaminidase secreted into the supernatant on the substrate p-nitrophenyl-N-actyl-β-O-glucosamine. All degranulation assays were repeated in at least triplicates. In all experiments the IgE concentration was 1 μg/mL.