title: A real-time fluorometric method for the simultaneous detection of cell death type and rate.
abstract: 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 (FAN) 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 (SYTOX 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 ∼2 h.
authors.affiliation: Molecular Signaling and Cell Death Unit, VIB Inflammation Research Center, Ghent, Belgium.|Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium.
authors.forename: Sasker
authors.identifier: 0000-0002-8385-2545
authors.initials: S
authors.lastname: Grootjans
authors.affiliation: Molecular Signaling and Cell Death Unit, VIB Inflammation Research Center, Ghent, Belgium.|Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium.
authors.forename: Behrouz
authors.initials: B
authors.lastname: Hassannia
authors.affiliation: Molecular Signaling and Cell Death Unit, VIB Inflammation Research Center, Ghent, Belgium.|Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium.
authors.forename: Iris
authors.initials: I
authors.lastname: Delrue
authors.affiliation: Molecular Signaling and Cell Death Unit, VIB Inflammation Research Center, Ghent, Belgium.|Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium.
authors.forename: Vera
authors.initials: V
authors.lastname: Goossens
authors.affiliation: Molecular Signaling and Cell Death Unit, VIB Inflammation Research Center, Ghent, Belgium.|Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium.
authors.forename: Bartosz
authors.initials: B
authors.lastname: Wiernicki
authors.affiliation: Molecular Signaling and Cell Death Unit, VIB Inflammation Research Center, Ghent, Belgium.|Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium.
authors.forename: Yves
authors.initials: Y
authors.lastname: Dondelinger
authors.affiliation: Molecular Signaling and Cell Death Unit, VIB Inflammation Research Center, Ghent, Belgium.|Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium.
authors.forename: Mathieu J M
authors.initials: MJ
authors.lastname: Bertrand
authors.affiliation: Molecular Signaling and Cell Death Unit, VIB Inflammation Research Center, Ghent, Belgium.|Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium.
authors.forename: Dmitri V
authors.initials: DV
authors.lastname: Krysko
authors.affiliation: Gnomixx, Ghent, Belgium.
authors.forename: Marnik
authors.initials: M
authors.lastname: Vuylsteke
authors.affiliation: Molecular Signaling and Cell Death Unit, VIB Inflammation Research Center, Ghent, Belgium.|Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium.|Methusalem Program, Ghent University, Ghent, Belgium.
authors.forename: Peter
authors.identifier: 0000-0002-6669-8822
authors.initials: P
authors.lastname: Vandenabeele
authors.affiliation: Molecular Signaling and Cell Death Unit, VIB Inflammation Research Center, Ghent, Belgium.|Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium.
authors.forename: Tom
authors.initials: T
authors.lastname: Vanden Berghe
mesh_terms: D000818:Animals; D016923:Cell Death; D002460:Cell Line; D005470:Fluorometry / Q000379:methods*; D006801:Humans; D051379:Mice; D013194:Staining and Labeling; D013997:Time Factors