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Novel integrated microdialysis-amperometric system for in vitro detection of dopamine secreted from PC12 cells: design, construction, and validation

Migheli, Rossana and Puggioni, Giulia and Dedola, Sonia and Rocchitta, Gaia Giovanna Maria and Calia, Giammario and Bazzu, Gianfranco and Esposito, Giovanni and Lowry, John P. and O'Neill, Robert D. and Desole, Maria Speranza and Miele, Egidio and Serra, Pier Andrea (2008) Novel integrated microdialysis-amperometric system for in vitro detection of dopamine secreted from PC12 cells: design, construction, and validation. Analytical Biochemistry, Vol. 380 (2), p. 323-330. ISSN 0003-2697. Article.

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DOI: 10.1016/j.ab.2008.05.050

Abstract

A novel dual channel in vitro apparatus, derived from a previously described design, has been coupled with dopamine (DA) microsensors for the flow-through detection of DA secreted from PC12 cells. The device, including two independent microdialysis capillaries, was loaded with a solution containing PC12 cells while a constant phosphate-buffered saline (PBS) medium perfusion was carried out using a dual channel miniaturized peristaltic pump. One capillary was perfused with normal PBS, whereas extracellular calcium was removed from extracellular fluid of the second capillary. After a first period of stabilization and DA baseline recording, KCl (75 mM) was added to the perfusion fluid of both capillaries. In this manner, a simultaneous "treatment-control" experimental design was performed to detect K+-evoked calcium-dependent DA secretion. For this purpose, self-referencing DA microsensors were developed, and procedures for making, testing, and calibrating them are described in detail. The electronic circuitry was derived from previously published schematics and optimized for dual sensor constant potential amperometry applications. The microdialysis system was tested and validated in vitro under different experimental conditions, and DA secretion was confirmed by high-performance liquid chromatography with electrochemical detection (HPLC-EC). PC12 cell viability was quantified before and after each experiment. The proposed apparatus serves as a reliable model for studying the effects of different drugs on DA secretion through the direct comparison of extracellular DA increase in treatment-control experiments performed on the same initial PC12 cell population.

Item Type:Article
ID Code:1359
Status:Published
Refereed:Yes
Uncontrolled Keywords:Microdialysis, dopamine, PC12, secretion, microsensor, amperometry, potentiostat
Subjects:Area 05 - Scienze biologiche > BIO/14 Farmacologia
Divisions:001 Università di Sassari > 01 Dipartimenti > Neuroscienze, scienze materno infantili
Publisher:Academic Press inc. Elsevier Science
ISSN:0003-2697
Publisher Policy:© 2008 Elsevier
Deposited On:18 Aug 2009 10:04

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