A real-time fluorometric method for the simultaneous detection of cell death type and rate

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.

[1]  R. McPherson,et al.  Correction: Galactose Enhances Oxidative Metabolism and Reveals Mitochondrial Dysfunction in Human Primary Muscle Cells , 2012, PLoS ONE.

[2]  S. Maeda,et al.  IVbteA Modified Colorimetric MTT Assay Adapted fbr Application to Proliferation and Cytotoxicity Assays , 2018 .

[3]  P. Vandenabeele,et al.  Necroptosis and its role in inflammation , 2015, Nature.

[4]  Elin Lindhagen,et al.  The fluorometric microculture cytotoxicity assay , 2008, Nature Protocols.

[5]  I. Wilson,et al.  Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. , 2000, European journal of biochemistry.

[6]  L. de Ridder,et al.  Phosphatidylserine exposure during early primary necrosis (oncosis) in JB6 cells as evidenced by immunogold labeling technique , 2004, Apoptosis.

[7]  Matthew E. Welsch,et al.  Regulation of Ferroptotic Cancer Cell Death by GPX4 , 2014, Cell.

[8]  K. Kersse,et al.  RIPK1 promotes death receptor-independent caspase-8-mediated apoptosis under unresolved ER stress conditions , 2014, Cell death & disease.

[9]  W. Fiers,et al.  Death receptor-induced apoptotic and necrotic cell death: differential role of caspases and mitochondria , 2001, Cell Death and Differentiation.

[10]  M. Glicksman,et al.  Identifying Druglike Inhibitors of Myelin-Reactive T Cells by Phenotypic High-Throughput Screening of a Small-Molecule Library , 2007, Journal of biomolecular screening.

[11]  F. Chan,et al.  Phosphorylation-Driven Assembly of the RIP1-RIP3 Complex Regulates Programmed Necrosis and Virus-Induced Inflammation , 2009, Cell.

[12]  Carlo Riccardi,et al.  Analysis of apoptosis by propidium iodide staining and flow cytometry , 2006, Nature Protocols.

[13]  J. Silke,et al.  Ars Moriendi; the art of dying well – new insights into the molecular pathways of necroptotic cell death , 2014, EMBO reports.

[14]  Na Zhang,et al.  RIP3, an Energy Metabolism Regulator That Switches TNF-Induced Cell Death from Apoptosis to Necrosis , 2009, Science.

[15]  T. Vanden Berghe,et al.  Disruption of HSP90 Function Reverts Tumor Necrosis Factor-induced Necrosis to Apoptosis* , 2003, The Journal of Biological Chemistry.

[16]  Peter Vandenabeele,et al.  Determination of apoptotic and necrotic cell death in vitro and in vivo. , 2013, Methods.

[17]  S. Cook,et al.  EndoG Links Bnip3-Induced Mitochondrial Damage and Caspase-Independent DNA Fragmentation in Ischemic Cardiomyocytes , 2011, PloS one.

[18]  P. Vandenabeele,et al.  Methods to Study and Distinguish Necroptosis , 2014 .

[19]  W. Fiers,et al.  Tumour necrosis factor-induced necrosis versus anti-Fas-induced apoptosis in L929 cells. , 1997, Cytokine.

[20]  Maryam Rashidi,et al.  RIPK3 promotes cell death and NLRP3 inflammasome activation in the absence of MLKL , 2015, Nature Communications.

[21]  N. Plesnila,et al.  Role of apoptosis inducing factor (AIF) for hippocampal neuronal cell death following global cerebral ischemia in mice , 2011, Neuroscience Letters.

[22]  P. Vandenabeele,et al.  Regulated necrosis: the expanding network of non-apoptotic cell death pathways , 2014, Nature Reviews Molecular Cell Biology.

[23]  T. Mosmann.  Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. , 1983, Journal of immunological methods.

[24]  Tao Wang,et al.  Receptor Interacting Protein Kinase-3 Determines Cellular Necrotic Response to TNF-α , 2009, Cell.

[25]  N. Thornberry,et al.  Determination of caspase specificities using a peptide combinatorial library. , 2000, Methods in enzymology.

[26]  M. Poot,et al.  Bacterial viability and antibiotic susceptibility testing with SYTOX green nucleic acid stain , 1997, Applied and environmental microbiology.

[27]  M. Bertrand,et al.  MLKL compromises plasma membrane integrity by binding to phosphatidylinositol phosphates. , 2014, Cell reports.

[28]  M. Bertrand,et al.  NF-κB-Independent Role of IKKα/IKKβ in Preventing RIPK1 Kinase-Dependent Apoptotic and Necroptotic Cell Death during TNF Signaling. , 2015, Molecular cell.

[29]  P. Vandenabeele,et al.  Tipping the balance between necrosis and apoptosis in human and murine cells treated with interferon and dsRNA , 2002, Cell Death and Differentiation.

[30]  Alexei Degterev,et al.  Identification of RIP1 kinase as a specific cellular target of necrostatins. , 2008, Nature chemical biology.

[31]  M. Bertrand,et al.  Molecular crosstalk between apoptosis, necroptosis, and survival signaling , 2015, Molecular & cellular oncology.

[32]  P. Vandenabeele,et al.  RIPK3 contributes to TNFR1-mediated RIPK1 kinase-dependent apoptosis in conditions of cIAP1/2 depletion or TAK1 kinase inhibition , 2013, Cell Death and Differentiation.

[33]  James O. Jones,et al.  Suppression of Antitumor Immunity by Stromal Cells Expressing , 2022 .

[34]  M. Bertrand,et al.  Vaccination with Necroptotic Cancer Cells Induces Efficient Anti-tumor Immunity. , 2016, Cell reports.

[35]  Wim Declercq,et al.  Differential Signaling to Apoptotic and Necrotic Cell Death by Fas-associated Death Domain Protein FADD* , 2004, Journal of Biological Chemistry.

[36]  J. Verspurten,et al.  Proteome-wide Substrate Analysis Indicates Substrate Exclusion as a Mechanism to Generate Caspase-7 Versus Caspase-3 Specificity* , 2009, Molecular & Cellular Proteomics.

[37]  Yide Mei,et al.  Cleavage of RIP3 inactivates its caspase-independent apoptosis pathway by removal of kinase domain. , 2007, Cellular signalling.

[38]  T. Vanden Berghe,et al.  Necroptosis, in vivo detection in experimental disease models. , 2014, Seminars in cell & developmental biology.

[39]  P. Vandenabeele,et al.  Depletion of RIPK3 or MLKL blocks TNF-driven necroptosis and switches towards a delayed RIPK1 kinase-dependent apoptosis , 2014, Cell Death and Disease.

[40]  P. Vandenabeele,et al.  Necroptosis, necrosis and secondary necrosis converge on similar cellular disintegration features , 2010, Cell Death and Differentiation.

[41]  P. Vandenabeele,et al.  The Potential Role of Necroptosis in Diseases , 2014 .

[42]  L. Yielding,et al.  Structure-function characterization of phenanthridinium compounds as mutagens in Salmonella. , 1983, Mutation research.