Dataset Information

Robust high-throughput kinetic analysis of apoptosis with real-time high-content live-cell imaging.

ABSTRACT: Quantitative and kinetic analyses of apoptotic cell death are integral components of exploring cell biology, measuring cellular stress responses, and performing high-throughput genomic/RNAi/drug screens. Here, we present a detailed method that integrates robust kinetic real-time high-content imaging with Annexin V labelling to provide a highly sensitive, accurate, simple and zero-handling approach to quantify extrinsic and intrinsic inducers of apoptosis. The sensitivity of this non-toxic method outperforms previous high-throughput methodologies using viability dyes or caspase-activation reporters. This method also incorporates a multiplex adaptation to integrate variability in cell number due to treatment-induced proliferation changes and the detachment of dying cells. Compared to Annexin V detection by flow cytometry, this method is 10-fold more sensitive, eliminates extensive sample handling and processing, and provides real-time kinetics of apoptosis at both single-cell and population-level resolutions.

SUBMITTER: Gelles JD

PROVIDER: S-EPMC5261025 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Robust high-throughput kinetic analysis of apoptosis with real-time high-content live-cell imaging.

Gelles Jesse D JD Chipuk Jerry Edward JE

Cell death & disease 20161201 12

Quantitative and kinetic analyses of apoptotic cell death are integral components of exploring cell biology, measuring cellular stress responses, and performing high-throughput genomic/RNAi/drug screens. Here, we present a detailed method that integrates robust kinetic real-time high-content imaging with Annexin V labelling to provide a highly sensitive, accurate, simple and zero-handling approach to quantify extrinsic and intrinsic inducers of apoptosis. The sensitivity of this non-toxic method ...[more]

PMID: 27906190

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Project description:Cell-surface glycans are attractive targets for molecule imaging due to their reflection of cellular processes associated with development and disease progression. In this paper, we describe the design, synthesis, and biological application of a new phosphine probe for real-time imaging of cell-surface glycans using bioluminescence. To accomplish this goal, we took advantage of the bioorthogonal chemical reporter technique. This strategy uses a two-step labeling procedure in which an unnatural sugar analogue containing a functional handle is (1) incorporated into sugar-bearing proteins via the cell's own biosynthetic machinery and then (2) detected with an exogenously added probe. We designed phosphine-luciferin reagent 1 to activate bioluminescence in response to Staudinger ligation with azide-labeled glycans. We chose to use a phosphine probe because, despite their slow reaction kinetics, they remain the best-performing reagents for tagging azidosugars in mice. Given the sensitivity and negligible background provided by bioluminescence imaging (BLI), we reasoned that 1 might be able to overcome some of the limitations encountered with fluorescent phosphine probes. In this work, we synthesized the first phosphine-luciferin probe for use in real-time BLI and demonstrated that azide-labeled cell-surface glycans can be imaged with 1 using concentrations as low as single digit nanomolar and times as little as 5 min, a feat that cannot be matched by any previous fluorescent phosphine probes. Even though we have only demonstrated its use in visualizing glycans, it can be envisioned that this probe could also be used for bioluminescence imaging of any azide-containing biomolecule, such as proteins and lipids, since azides have been previously incorporated into these molecules. The phosphine-luciferin probe is therefore poised for many applications in real-time imaging in cells and whole animals. These studies are currently in progress in our laboratory.

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Dataset Information

Robust high-throughput kinetic analysis of apoptosis with real-time high-content live-cell imaging.

Publications

Robust high-throughput kinetic analysis of apoptosis with real-time high-content live-cell imaging.

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