A convenient, high-throughput method for enzyme-luminescence detection of dopamine released from PC12 cells.
This protocol represents a novel enzyme-luminescence method to detect dopamine sensitively and rapidly with high temporal resolution. In principle, dopamine is first oxidized with tyramine oxidase to produce H(2)O(2), and then the produced H(2)O(2) reacts with luminol to generate chemiluminescence in the presence of horseradish peroxidase (POD). We applied this method successfully to perform real-time monitoring of dopamine release from PC12 cells using a luminescence plate reader upon stimulation with several drugs (e.g., acetylcholine, bradykinin). The results indicated that the dopamine release from PC12 cells was modulated by these drugs in a way similar to that found by using several conventional analytical techniques, such as HPLC-electrochemical detector (ECD). Unlike other assays, this assay technique is simple, rapid, highly sensitive and thus useful for assessment of effects of drugs on the nervous system. The dopamine release assay takes only < or =1 h once reagent setup and culture plates' preparation are finished.
Shinohara
Hiroaki
H
Field of Life, Information and System Sciences, Graduate School of Science and Engineering for Research, University of Toyama, Toyama, Japan. hshinoha@eng.u-toyama.ac.jp
Wang
Feifei
F
Hossain
S M Zakir
SM
D000109:Acetylcholine; D000818:Animals; D001920:Bradykinin; D004298:Dopamine / Q000737:chemistry / Q000378:metabolism*; D049449:Luminescence; D008163:Luminescent Measurements / Q000379:methods*; D008165:Luminol / Q000737:chemistry; D015394:Molecular Structure; D016716:PC12 Cells; D051381:Rats
D008165:Luminol; D000109:Acetylcholine; D001920:Bradykinin; D004298:Dopamine