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  • Monitoring apoptosis and neuronal degeneration by real-time detection of phosphatidylserine externalization using a polarity-sensitive indicator of viability and apoptosis.

Monitoring apoptosis and neuronal degeneration by real-time detection of phosphatidylserine externalization using a polarity-sensitive indicator of viability and apoptosis.

Nature protocols (2010-07-31)
Yujin E Kim, Jeannie Chen, Ralf Langen, Jonah R Chan
RESUMEN

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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Sigma-Aldrich
Potassium phosphate monobasic, powder, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, ≥99.0%
Sigma-Aldrich
Sodium phosphate dibasic, BioXtra, ≥99.0%
Sigma-Aldrich
Ethylenediaminetetraacetic acid disodium salt dihydrate, suitable for electrophoresis, for molecular biology, 99.0-101.0% (titration)
Sigma-Aldrich
Calcium chloride dihydrate, for molecular biology, ≥99.0%
Sigma-Aldrich
1,2-Diacyl-sn-glycero-3-phospho-L-serine, ≥97% (TLC), from bovine brain, amorphous powder
Sigma-Aldrich
L-α-Phosphatidylcholine, egg yolk, Type XVI-E, ≥99% (TLC), lyophilized powder