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REPORT

CELLULAR METABOLISM

A novel method using fluorescence microscopy for real-time assessment of ATP release from individual cells

¹Department of Surgery, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts; and Departments of ²Surgery and ³Pharmacology and Medicine, and ⁴Biomedical Sciences Graduate Program, University of California, San Diego, California

Submitted 26 July 2007 ; accepted in final form 9 August 2007

ABSTRACT

Many cell types release ATP in response to mechanical or biochemical stimulation. The mechanisms responsible for this release, however, are not well understood and may differ among different cell types. In addition, there are numerous difficulties associated with studying the dynamics of ATP release immediately outside the cell membrane. Here, we report a new method that allows the visualization and quantification of ATP release by fluorescence microscopy. Our method utilizes a two-enzyme system that generates NADPH when ATP is present. NADPH is a fluorescent molecule that can be visualized by fluorescence microscopy using an excitation wavelength of 340 nm and an emission wavelength of 450 nm. The method is capable of detecting ATP concentrations <1 µM and has a dynamic range of up to 100 µM. Using this method, we visualized and quantified ATP release from human polymorphonuclear leukocytes and Jurkat T cells. We show that upon cell stimulation, the concentrations of ATP can reach levels of up to 80 µM immediately outside of the cell membrane. This new method should prove useful for the study of the mechanisms of release and functional role of ATP in various cell systems, including individual cells.

purinergic signaling

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