| Monitoring apoptosis and neuronal degeneration by real-time detection of phosphatidylserine externalization using a polarity-sensitive indicator of viability and apoptosis. | |
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MedLine Citation:
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PMID: 20671723 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Applications for noninvasive real-time imaging of apoptosis and neuronal degeneration are hindered by technical limitations in imaging strategies and by existing probes. Monitoring the progression of a cell through apoptosis could provide valuable insight into the temporal events that initiate cell death as well as the potential for rescue of apoptotic cells. We engineered an annexin-based biosensor to function as a polarity-sensitive indicator for viability and apoptosis (known as pSIVA) by binding to externalized phosphatidylserine (PS) exposed on apoptotic cell membranes. Constructed from a structure-based design strategy, pSIVA fluoresces only when bound to PS and remains effectively undetectable in solution. In this paper, we describe protocols for the design, expression, purification and labeling of pSIVA as well as for its application in time-lapse imaging of degenerating neurons in culture; the entire protocol can be completed in 2 weeks. The primary advantage of this method is the flexibility to use pSIVA, in combination with other probes and without perturbing experimental conditions, to explore the cellular mechanisms involved in apoptosis and degeneration in real time. |
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Authors:
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Yujin E Kim; Jeannie Chen; Ralf Langen; Jonah R Chan |
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Publication Detail:
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Type: Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't Date: 2010-07-08 |
Journal Detail:
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Title: Nature protocols Volume: 5 ISSN: 1750-2799 ISO Abbreviation: Nat Protoc Publication Date: 2010 Aug |
Date Detail:
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Created Date: 2010-07-30 Completed Date: 2010-12-02 Revised Date: 2011-01-10 |
Medline Journal Info:
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Nlm Unique ID: 101284307 Medline TA: Nat Protoc Country: England |
Other Details:
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Languages: eng Pagination: 1396-405 Citation Subset: IM |
Affiliation:
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Department of Biochemistry and Molecular Biology, Zilkha Neurogenetic Institute at the Keck School of Medicine, University of Southern California, Los Angeles, California, USA. |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Animals Annexins / analysis, chemistry Apoptosis* Biosensing Techniques* Calcium / chemistry Cell Culture Techniques Cell Membrane / metabolism, ultrastructure Cell Survival Culture Media Escherichia coli / genetics Female Ganglia, Spinal / cytology, metabolism, ultrastructure Microscopy, Fluorescence / methods Nerve Degeneration / pathology* Neurons / cytology*, metabolism Oxadiazoles / analysis Phosphatidylserines / analysis* Rats Rats, Sprague-Dawley |
| Grant Support | |
ID/Acronym/Agency:
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EY12155/EY/NEI NIH HHS; R01 EY012155-14/EY/NEI NIH HHS |
| Chemical | |
Reg. No./Substance:
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0/Annexins; 0/Culture Media; 0/Oxadiazoles; 0/Phosphatidylserines; 67013-48-3/4-(N-(iodoacetoxy)ethyl-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole; 7440-70-2/Calcium |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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