Automated live cell imaging of green fluorescent protein degradation in individual fibroblasts.

Automated live cell imaging of green fluorescent protein degradation in individual fibroblasts. Research Abstract Details 

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Automated live cell imaging of green fluorescent protein degradation in individual fibroblasts. Abstract Text:

Michael Halter,Alex Tona,Kiran Bhadriraju,Anne L Plant,John T Elliott,Michael Halter,Alex Tona,Kiran Bhadriraju,Anne L Plant,John T Elliott,

To accurately interpret the data from fluorescent proteins as reporters of gene activation within living cells, it is important to understand the kinetics of the degradation of the reporter proteins. We examined the degradation kinetics over a large number (>1,000) of single, living cells from a clonal population of NIH3T3 fibroblasts that were stably transfected with a destabilized, enhanced green fluorescent protein (eGFP) reporter driven by the tenascin-C promoter. Data collection and quantification of the fluorescence protein within a statistically significant number of individual cells over long times (14 h) by automated microscopy was facilitated by culturing cells on micropatterned arrays that confined their migration and allowed them to be segmented using phase contrast images. To measure GFP degradation rates unambiguously, protein synthesis was inhibited with cycloheximide. Results from automated live cell microscopy and image analysis indicated a wide range of cell-to-cell variability in the GFP fluorescence within individual cells. Degradation for this reporter was analyzed as a first order rate process with a degradation half-life of 2.8 h. We found that GFP degradation rates were independent of the initial intensity of GFP fluorescence within cells. This result indicates that higher GFP abundance in some cells is likely due to higher rates of gene expression, because it is not due to systematically lower rates of protein degradation. The approach described in this study will assist the quantification and understanding of gene activity within live cells using fluorescent protein reporters.

Automated live cell imaging of green fluorescent protein degradation in individual fibroblasts. Publishing Authors By Initials

M Halter,A Tona,K Bhadriraju,AL Plant,JT Elliott,M Halter,A Tona,K Bhadriraju,AL Plant,JT Elliott,

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Automated live cell imaging of green fluorescent protein degradation in individual fibroblasts. Journal Published:

PUBLICATION TYPE: Journal Article

Journal: Cytometry. Part A : the journal of the Internation

VOLUME: 71

Page Numbers: 827-34

Journal Abbreviation: Cytometry A

ISSN: 1552-4922

DAY: 26

MONTH: Oct

YEAR: 2007

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LANGUAGE: eng

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AFFILIATION: Cell and Tissue Measurements Group, Biochemical Science Division, Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA. michael.halter@nist.gov

Country: United States

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MEDLINETA: Cytometry A

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