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Am J Physiol Cell Physiol 284: C1083-C1089, 2003. First published October 23, 2002; doi:10.1152/ajpcell.00276.2002
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Vol. 284, Issue 4, C1083-C1089, April 2003

Live cell imaging using confocal microscopy induces intracellular calcium transients and cell death

Martin M. Knight, Susan R. Roberts, David A. Lee, and Dan L. Bader

Interdisciplinary Research Centre, Biomedical Materials and Medical Engineering Division, Department of Engineering, Queen Mary University of London, London E1 4NS, United Kingdom

Isolated chondrocytes stained with fluo 4-AM and visualized using standard confocal microscopy techniques exhibited Ca2+ transients and oscillations. Decreasing the power of the laser light decreased the percentage of cells exhibiting these Ca2+ signals. Treatment with the antioxidant ascorbate reduced the Ca2+ response, suggesting that it was mediated by light-induced release of reactive oxygen species (ROS). Cell viability 24 h after the 1-h confocal imaging period was ~90% for cells that were neither fluorescently stained nor subjected to laser excitation. By contrast, fluorescently stained cells imaged for 1 h exhibited greatly reduced viability. Treatment with ascorbate reduced the level of cell death, suggesting that the effect was mediated by release of exogenous ROS associated with the interaction of light and the fluorochrome. Ca2+ oscillations were not always associated with cell death, suggesting that separate light-sensitive pathways mediate the two processes. Light-activated Ca2+ signaling may trigger alterations in numerous cell processes and thereby represent an important and hitherto overlooked artifact in fluorescent microscopy of viable cells.

photoactivation; free radicals; reactive oxygen species; oxygen; chondrocyte


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