Nanoscale Patterning of Antigen on Silicon Substrate to Examine Mast Cell Activation
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Nanoscale Patterning of Antigen on Silicon Substrate to Examine Mast Cell Activation Reid N. Orth1*, Min Wu2*, Theodore G. Clark3, David A. Holowka2, Barbara A. Baird2, and Harold G. Craighead1 1 School of Applied and Engineering Physics, 2Department of Chemistry and Chemical Biology, Cornell University, 3Department of Microbiology and Immunology Ithaca, NY 14853 ABSTRACT Rat Basophilic Leukemia (RBL) cells are immobilized and stimulated on micro- and nanometer scale patterns of supported lipid bilayers. The patterns are realized as the photolithographically patterned polymer is mechanically peeled away in one contiguous piece in solution. The 0.36 µm2 to 4,489 µm2 patches can contain both fluorescent lipids and lipid-linked antigen and provide a synthetic biological substrate for analysis of cell surface receptor-mediated events. 100-nm unilamellar lipid vesicles spread to form a supported lipid bilayer on a thermally oxidized silicon surface as confirmed by fluorescence recovery after photobleaching (FRAP). Aggregation of fluorescently labeled receptors is observed as their coincidence with the patterned antigen. Cell morphology is analyzed with scanning electron microscopy (SEM). Thus, a novel method has been developed for patterning antigen, capturing and immobilizing cells via specific receptors, and spatially controlling antigenic stimulus on the nanoscale. INTRODUCTION Dinitrophenyl-conjugated lipids have been patterned on the micro- and nanometer scale to spatially control the stimulation of specific immunoreceptors on RBL mast cells. This work was motivated by previous research to elucidate the cascade of events from the initial receptor ligand interaction through cellular activation. Typically, many membrane-bound molecules work collectively to achieve specific cell-cell and cell-substrate recognition. The immunological synapse formation between antigen receptors on T cells (TCR) and antigen presenting cells is an example of the complex interaction initiating T cell activation in many immune responses [1]. IgE receptors (IgE-FcεRI) on mast cells are structurally and functionally similar to TCR and they are mainly involved in the allergic immune response and related inflammatory diseases. IgE are soluble, Y-shaped antibodies with binding sites for antigen in each of two segments. The third segment binds tightly to FcεRI on the mast cell surface, thereby becoming part of the receptor and effectively sensitizing the cell to the specific antigen. Cross-linking of IgE-FcεRI by bi- or multivalent antigens initiates transmembrane signaling which leads to exocytosis of secretory granules containing histamine and other inflammatory mediators. Mast cells are longterm residents of vascularized tissues and have specific roles in acute, ‘late phase’ and chronic aspects of adaptive or pathological IgE-associated acquired immune responses [2]. Recent research on immunoreceptor signaling has begun to focus on the initial stages involving specialized membrane domains, commonly called lipid rafts. In the mast cell system, f
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