ALBERT

Description: Inlive cellimaging of fluorescent proteins, phototoxicity of the excitation light can be problematical. Cell death is obvious, but reduced cell viability can make the interpretation of observations error prone. We characterized the phototoxic consequences of 488 and 546nm light on untransformed human cells and tested methodsthat have or could be used to alleviate photodamage.Unlabeled RPE1 cells were given single 0.5 to 2.5 minute irradiations in early G1 from a mercury arc lamp on a fluorescence microscope. 488nm lightproduced a dose dependent decrease in the percentage of cells that progressed to mitosis, slowing of the cell cycle for some of those entering mitosis, and a ∼12% incidence of cell deathfor the highest dose. For 546nm light we found a 10-15% reduction in the percentage of cells entering mitosis, no strong dose dependency, and a ∼2% incidence of cell deathfor the longest irradiations. For cells expressing GFP-centrin1 or mCherry-centrin1, fewer entered mitosis for each dose than unlabeled cells. For constant total dose 488nm light irradiations of unlabeled cells, reducing the intensity tenfold or spreading the exposures out as a series of 10 second pulses at 1 minute intervals produced a minor and not consistent improvement in the percentage of cells entering mitosis. Reducing oxidative processes, by culturing at ∼3% oxygen or adding the reducing agent Troloxnoticeably increased the fraction of cells entering mitosis. Thus, for long term imaging there can be value to using RFP constructs and for GFP tagged proteins reducing oxidative processes. This article is protected by copyright. All rights reserved