期刊
LAB ON A CHIP
卷 8, 期 1, 页码 161-169出版社
ROYAL SOC CHEMISTRY
DOI: 10.1039/b715308m
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- NATIONAL CENTER FOR RESEARCH RESOURCES [S10RR022578] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE [R01NS048826] Funding Source: NIH RePORTER
- NCRR NIH HHS [S10 RR022578, 1S10RR022578] Funding Source: Medline
- NINDS NIH HHS [R01 NS048826-01, NS048826, R01 NS048826] Funding Source: Medline
Photorelease of caged Ca2+ is a uniquely powerful tool to study the dynamics of Ca2+-triggered exocytosis from individual cells. Using photolithography and other microfabrication techniques, we have developed transparent microchip devices to enable photorelease of caged Ca2+, together with electrochemical detection of quantal catecholamine secretion from individual cells or cell arrays as a step towards developing high-throughput experimental devices. A 100 nm thick transparent indium-tin-oxide (ITO) film was sputter-deposited onto glass coverslips, which were then patterned into 24 cell-sized working electrodes (similar to 20 mu m by 20 mu m). We loaded bovine chromaffin cells with acetoxymethyl (AM) ester derivatives of the Ca2+ cage NP-EGTA and Ca2+ indicator dye fura-4F, then transferred these cells onto the working ITO electrodes for amperometric recordings. Upon flash photorelease of caged Ca2+, a uniform rise of [Ca2+](i) within the target cell leads to quantal release of oxidizable catecholamines measured amperometrically by the underlying ITO electrode. We observed a burst of amperometric spikes upon rapid elevation of [Ca2+](i) and a '' priming '' effect of sub-stimulatory [Ca2+](i) on the response of cells to subsequent [Ca2+](i) elevation, similar to previous reports using different techniques. We conclude that UV photolysis of caged Ca2+ is a suitable stimulation technique for higher-throughput studies of Ca2+-dependent exocytosis on transparent electrochemical microelectrode arrays.
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