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A convenient, high-throughput method for enzyme-luminescence detection of dopamine released from PC12 cells

Nature Protocols volume 3, pages 1639–1644 (2008)Cite this article

Abstract

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 H2O2, and then the produced H2O2 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 ≤1 h once reagent setup and culture plates' preparation are finished.

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Figure 1: Schematic representation of the detection principle of the enzyme-catalyzed chemiluminescence assay for dopamine.
Figure 2: Dopamine-releasing mechanisms in an excited PC12 cell upon stimulation with both acetylcholine (ACh) and bradykinin (BK).
Figure 3: Dose-dependent effects of various concentrations of ACh (0–500 μM) on the release of dopamine from PC12 cells.
Figure 4: Dose-dependent effects of various concentrations of BK (0–250 nM) on the dopamine release from PC12 cells.

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Acknowledgements

This work was supported by a grant of Toyama-Medical Bio Cluster project, MEXT in Japan.

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Authors and Affiliations

  1. Field of Life, Information and System Sciences, Graduate School of Science and Engineering for Research, University of Toyama, 3190 Gofuku, Toyama, 930-8555, Japan

    Hiroaki Shinohara

  2. Course of Biological Information Systems, Graduate School of Innovative Life Science for Education, University of Toyama, 3190 Gofuku, Toyama, 930-8555, Japan

    Hiroaki Shinohara

  3. Department of Neurological Surgery, Graduate School of Medicine, Okayama University, 2-5-1, Shikata-cho, Okayama, 700-8558, Japan

    Feifei Wang

  4. Course of Advanced Nano and Biosciences, Graduate School of Innovative Life Science for Education, University of Toyama, 3190 Gofuku, Toyama, 930-8555, Japan

    S M Zakir Hossain

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  1. Hiroaki Shinohara
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Correspondence to Hiroaki Shinohara or S M Zakir Hossain.

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Shinohara, H., Wang, F. & Hossain, S. A convenient, high-throughput method for enzyme-luminescence detection of dopamine released from PC12 cells. Nat Protoc 3, 1639–1644 (2008). https://doi.org/10.1038/nprot.2008.158

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