Dnp25 bsa

MEM was purchased from Life Technologies (Grand Island, NY). Alexa⁶⁴⁷-labeled anti-Tac IgG was purchased from BioLegend (San Diego, CA) and anti-mouse IgG was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Amaxa Nucleofector and Solution L were purchased from Lonza (Basel, Switzerland). Fura-2AM was from Molecular Probes (Eugene, OR). DNP₂₅-BSA was from ThermoFisher Scientific (Waltham, MA; catalogue #A23018) and anti-DNP-IgE was affinity-purified from ascites (Covance, Denver, PA) according to the methods of Liu et al. (1980) (link). Alexa⁶⁴⁷-labeled IgE was prepared using Alexa⁶⁴⁷ NHS Ester (succinimidyl ester; ThermoFisher Scientific).

MEM media was purchased from Life Technologies (Grand Island, NY). G418 Disulfate was obtained from Caisson Labs (G030-5GM). Fura-2AM was purchased from Molecular Probes (Eugene, OR). DNP₂₅-BSA was from ThermoFisher Scientific (Waltham, MA; catalogue #A23018), custom DF3 peptide synthesized by and purchased from Anaspec (Fremont, CA) and anti-DNP-IgE was affinity-purified from ascites (Covance, Denver, PA) according to the methods of Liu et al (28 ). AlexaFluor647- (Thermofisher Scientific #A-20006), Janelia Fluor 646- (Tocris #6148) and CF640R- (Biotium #92108) labeled IgE lots were prepared using NHS Ester conjugation. Antibodies used are listed as follows: anti-pY PY20/99 cocktail (SantaCruz; sc-508, sc-7020), anti-rabbit-HRP secondary (SantaCruz; sc-2004, Cell Signaling #7074), anti-FcεRIγ (EMD Millipore; 06–727), Goat anti-rat IgE (Abnova; PAB29749). Poly-L lysine was purchased from Sigma-Aldrich (P1524-100MG), 18:1 (Δ9-Cis) PC (DOPC) and 16:0 DNP Cap PE lipids were purchased from Avanti Polar Lipids (850375C and 810528C, respectively). DNP-Peg12-NHS ester was purchased from BroadPharm (BP-22397). All live-cell RBL imaging was performed in modified Hank’s buffered salt solution (additional 10 mM Hepes, 0.05% w/v BSA, 5.45 mM glucose, 0.88 mM MgSO₄, 1.79 mM CaCl₂, 16.67 mM NaHCO₃).

MEM was purchased from Life Technologies (Grand Island, NY). AlexaFlour647 (AF647)-labeled anti-Tac IgG was from BioLegend (San Diego, CA), and anti-mouse IgG was from Jackson ImmunoResearch Laboratories (West Grove, PA). Fura-2AM was from Molecular Probes (Eugene, OR). DNP₂₅-BSA was from Thermo Fisher Scientific (Waltham, MA; catalogue #A23018), and anti-DNP-IgE was affinity purified from ascites (Covance, Denver, PA) according to the methods of Liu et al. (1980) (link). AF647-labeled IgE was prepared using AlexaFluor647 NHS Ester (succinimidyl ester; Thermo Fisher Scientific). Janelia Fluor 646–labeled IgE (JF646) was prepared using Janelia Fluor 646 NHS Ester (Janelia Fluor 646 succinimidyl ester, Tocris; 6148). Antibodies used are as follows: anti-pY (4G10) was a gift from J. Cambier, (University of Colorado) anti–mouse-horseradish peroxidase (HRP) (Cell Signaling Technology; 7076S) and anti–rabbit-HRP secondary (Santa Cruz; sc-2004), Lyn (Abcam; ab71093), Src (Cell Signaling Technology; 2110), Lck (Antibodies-Online; ABIN4965391), Yes1 (R&D Systems; AF3205); Fyn (R&D Systems; MAB3574), SHIP1 (Santa Cruz; sc-136066), vinculin (Santa Cruz; sc-7F9), β-actin (Sigma; A2668), FcεRIγ (EMD Millipore; 06-727), goat anti-rat IgE (Abnova; PAB29749).