Real Time Detection of 1A-ARs Movement Stimulated by Phenylephrine in Single Living Cell

IM: To investigate the movement of 1A- adrenergic receptors( 1A-ARs) stimulated by agonist phenylephrine (PE) and the dynamics of receptors movement in real time in single living cells with millisecond resolution. METHODS: We labeled 1A-ARs using monoclonal anti-FLAG antibody and Cy3-conjugated Goat Anti-Mouse IgG and recorded the trajectory of their transport process in the living HEK293A cells stimulated by agonist phenylephrine (PE), then analysis the dynamic properties of them. RESULTS: The specific detection of 1A-ARs on the surface of living HEK293A- 1A cells was achieved. 1A-ARs internalize under stimulation of PE After the cells were stimulated with PE for 20 minutes, apparent co-localization was found between 1A-ARs and F-actins. And after 40 min stimulation of PE, trajectories of approximate linear motion in HEK293A- 1A cells were recorded, and the velocity of them were calculated. CONCLUSION: The specific labeling method on living cell surface provides a convenient means on real-time detection of the behavior of surface receptors. By this method we were able to specifically detect 1A-ARs, record the behavior of individual particles of receptors with 50ms exposure time in real time in single living cell.