High affinity IgE receptor diffusional dynamics measured by single quantum dot tracking in resting and activated cells

Abstract

The high affinity IgE receptor (FcεRI) of mast cells and basophils binds circulating IgE with sub‐nanomolar affinity. Crosslinking of IgE‐FcεRI complexes by multivalent allergen initiates a complex signaling cascade that leads to the release of inflammatory mediators. In order to study the dynamics of FcεRI signaling, we have generated two novel quantum dot (QD) based probes: monovalent QD‐IgE (binds FcεRI without crosslinking) and polyvalent 2,4‐dinitrophenol (DNP)‐QD (crosslinks DNP‐specific IgE). Through single QD tracking on the apical membrane of RBL‐2H3 cells, we observe a two‐fold decrease in the diffusion coefficient of IgE‐FcεRI complexes in the presence of saturating (dark) IgE‐FcεRI, suggesting homotypic interactions. By simultaneously tracking QD‐IgE and DNP‐QD, we observe that, at low allergen levels, DNP‐QD crosslinked receptors remain mobile, suggesting that receptor immobilization is not critical for signal initiation. Mathematical models incorporating these data show that slower diffusion rates of nearby receptors initiate clustering consistent with the distribution of immunolabeled receptors seen in electron micrographs. Taken together, these data indicate a mechanism through which FcεRI receptors maintain proximity and enhance the probability of signaling complex formation.

Synthetic Allergen Design Reveals the Significance of Moderate Affinity Epitopes in Mast Cell Degranulation

“…RBL cells were maintained as described previously. 38 For the degranulation assay, cells were plated at 0.5 × 10 6 cells/mL in a 96 well plate and were incubated overnight. The next morning, 1 μg/mL IgE was introduced to prime the cells at saturating concentrations.…”

“…RBL cells were kindly provided by Dr. Wilson (University of New Mexico). RBL cells were maintained as described previously . For the degranulation assay, cells were plated at 0.5 × 10 6 cells/mL in a 96 well plate and were incubated overnight.…”

Microfluidics on the fly: Inexpensive rapid fabrication of thermally laminated microfluidic devices for live imaging and multimodal perturbations of multicellular systems

“…40 The organs were dissected and cultured in Grace's Drosophila Medium (Sigma, G9771) supplemented with Bis-Tris (Sigma, B9754), Penn-Strep (Gibco, 15140122), and FBS (Gibco, 10438-026). 41 Rat basophil leukemia cells (ATCC, 2256) were grown in RBL-2H3 medium 42 and were seeded in the cell gradient-PETL (CG-PETL) microfluidic devices at a concentration of ∼3 × 10 6 cells/ml.…”

“…Gradients across the micro-organ were formed with a 0.1% solution of CellMask™ Deep Red (ThermoFisher, C10046) and with 10 μM Hoechst (Sigma, bisBenzimide H, B2883) dye dissolved into Graces Medium prepared according to Dye et al 41 Cell gradient experiments were performed with 0.05% v/v CellMask™, 10 μM Hoechst, and 0.1% v/v Sytox (ThermoFisher, S34862), 1% red food coloring (McCormick, 52100071077), dissolved in RBL-2H3 medium. 42 Imaging PETL microfluidic devices were loaded with culture medium according to the specific cell or organ culture methods detailed above. Cells were seeded using syringe pumps (Harvard Apparatus PicoPlus Elite) at a flow rate of 60 μl/min.…”