Imaging Localized Astrocyte ATP Release with Firefly Luciferase Beads Attached to the Cell Surface

摘要

Extracellular adenosine triphosphate (ATP) functions as a signaling molecule in many cell regulation processes. The traditional firefly luciferase assays measure the ATP release as a signal increase with time using a luminometer. Recently, advanced cell imaging techniques using charge-coupled device (CCD) cameras have enabled two-dimensional (2D) high-resolution detection providing both spatial and temporal information. Real-time imaging of ATP release from astrocyte cells has been reported. However, the observed chemiluminescence propagation wave reflects both ATP release and diffusion in the extracellular bulk solution. The dynamic ATP efflux at the cell surface could not be accurately measured. Hence, we constructed biotinylated fused firefly luciferase proteins, immobilized the proteins on 1 (mu)m beads, and attached the beads to the cell surface to detect ATP release from mechanically stimulated astrocyte cells. This novel detection method enables us to monitor the actual ATP concentration at the surface of single live cells. The localized ATP release was found to be prominent but lasted only <20 s, which is very different from the results obtained by free firefly luciferase detection.