A real-time fluorometric method for the simultaneous detection of cell death type and rate
| Autor: | Marnik Vuylsteke, Behrouz Hassannia, Mathieu J.M. Bertrand, Peter Vandenabeele, Vera Goossens, Dmitri V. Krysko, Sasker Grootjans, Yves Dondelinger, Iris Delrue, Bartosz Wiernicki, Tom Vanden Berghe |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Programmed cell death Time Factors Necrosis Fluorophore Necroptosis Biology General Biochemistry Genetics and Molecular Biology Cell Line Paraptosis Mice 03 medical and health sciences chemistry.chemical_compound medicine Animals Humans Fluorometry Cytotoxicity Cell Death Staining and Labeling Cell biology Chemistry 030104 developmental biology chemistry Cell culture Apoptosis medicine.symptom |
| Zdroj: | Nature protocols |
| ISSN: | 1750-2799 1754-2189 |
| Popis: | Several cell death assays have been developed based on a single biochemical parameter such as caspase activation or plasma membrane permeabilization. Our fluorescent apoptosis/necrosis (FANAN) assay directly measures cell death and distinguishes between caspase-dependent apoptosis and caspase-independent necrosis of cells grown in any multiwell plate. Cell death is monitored in standard growth medium as an increase in fluorescence intensity of a cell-impermeable dye (SYTOTOX Green) after plasma membrane disintegration, whereas apoptosis is detected through caspase-mediated release of a fluorophore from its quencher (DEVD-amc). The assay determines the normalized percentage of dead cells and caspase activation per condition as an end-point measurement or in real time (automated). The protocol can be applied to screen drugs, proteins or siRNAs for interference with cell death while simultaneously detecting cell death modality switching between apoptosis and necrosis. Initial preparation may take up to 5 d, but the typical hands-on time is similar to 2 h. |
| Databáze: | OpenAIRE |
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