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Localized Functional Chemical Stimulation of TE 671 Cells Cultured on Nanoporous Membrane by Calcein and Acetylcholine

Susanne Zibek ^*, Alfred Stett ^*, Peter Koltay , Min Hu , Roland Zengerle , Wilfried Nisch ^* and Martin Stelzle ^*

^* Natural and Medical Sciences Institute, D-72770 Reutlingen, Germany; and Institute of Microsystem Technology IMTEK, D-79110 Freiburg, Germany

Correspondence: Address reprint requests and inquiries to Dr. Martin Stelzle, Natural and Medical Sciences Institute, Tel.: 49-7121-5153075; Fax: 49-7121-5153062; E-mail: stelzle{at}nmi.de.

Acetylcholine sensitive TE 671 cells were cultured on nanoporous membranes and chemically stimulated by localized application of i), calcein-AM and ii), acetylcholine, respectively, onto the bottom face of the membrane employing an ink jet print head. Stimulus correlated response of cells was recorded by fluorescence microscopy with temporal and spatial resolution. Calcein fluorescence develops as a result of intracellular enzymatic conversion of calcein-AM, whereas Ca²⁺ imaging using fluo-4 dye was employed to visualize cellular response to acetylcholine stimulation. Using 25 pl droplets and substance concentration ranging from 10 µM to 1 mM on Nucleopore membranes with pore diameters between 50 nm and 1 µm, a resolution on the order of 50 µm was achieved.