Using our shear-free microdevice, we measured the polarized distribution of individual receptors at the GCs membrane in response to GABA gradients with well-defined mean concentration c, steepness ?c, and orientation. By characterizing the polarization kinetics and amplitude in these variable gradients, we determined the amplification and filtering properties of nerve GCs during GABA directional sensing. With computational modeling, we further connected the GC response to properties of the chemoreceptor lateral dynamics and signaling activity. Overall, our assay provides an integrative description of GCs as chemical sensing modules, from a molecular to a systems-level. Membrane microdevices were prepared by an adapted soft imprint lithography technique using the ultraviolet (UV) polymerizable <a href="http://www.selleckchem.com/products/SB-431542.html">SB431542
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in vitro</a> material NOA 81 (Norland Products, Cranbury, NJ). The fabrication process is detailed in (20). Briefly, a two-level polydimethylsiloxane (PDMS) stamp was made by PDMS replica molding of a photolithographed 80/160 ��m SU-8 mold (Microchem, Newton, MA). The surface of this replica was then passivated with a perfluoro silane (Sigma, St Louis, MO), and the PDMS invert replica was made by conventional molding. A 5?�� 5?mm2 membrane (Cyclopore, ?20 ��m thickness, 400?nm hole diameter, Whatman, Maidstone, United Kingdom) was aligned and pressed between this two-level PDMS stamp and a PDMS flat layer. The space in between was capillary filled with NOA81, and <250 ��L of resin <a href="http://www.selleck.cn/products/azd5363.html">AZD5363</a>
are sufficient to form a 25?�� 50?mm2 microcircuit. When filling was complete, a uniform 365?nm illumination (Ligthningcure LC8, Hamamatsu Photonics, Hamamatsu City, Japan) for 15?s at 25 mW/cm2 through the PDMS was performed. A stiff micropatterned layer of NOA 81 with embedded membrane and channel network was obtained after PDMS removal. At the same time, a glass slide with drilled access holes (Sandblaster, Texas Airsonics, Corpus Christi, TX) and partially cured NOA 81 (365?nm, 15?s at 8 mW/cm2) was prepared. This slide was gently pressed over the previously prepared membrane-embedded layer and an additional UV illumination (15 s, 25 mW/cm2) was applied for irreversibly bonding the assembly. Microwells were prepared by the classical UV lithography technique. Briefly, a NOA 81 droplet was deposited onto a flat PDMS surface structured with 200 ��m high pillars, and a glass coverslip was placed on top to create a uniform layer. UV exposure (10 s, 8 mW/cm2) is performed across a transparency mask through the coverslip to form a 1?�� 1?mm2 chamber. After exposure, the coverslip with reticulated pattern was removed from the PDMS and noncross-linked NOA 81 was rinsed, first briefly with acetone and then extensively with ethanol. A final UV exposure was performed to fully cross-link the glue.