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

Nature Protocols volume 11, pages 1444–1454 (2016)Cite this article

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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.

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Figure 1: Principle and validation of the fluorescent apoptosis–necrosis assay.
Figure 2: Detection of apoptosis and necrosis by the FAN assay in different model systems.

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Acknowledgements

We acknowledge the following members of our laboratory: Q. Remijsen for his contribution to the initial development of the assay, and R. Roelandt, S. Martens, M. Aguileta and Y. Estornes for sharing data that were not included in the final manuscript. We acknowledge D. Fearon, University of Cambridge, for his kind gift of LL2-OVA cells. We are grateful to A. Bredan for editing the manuscript. Research in the Vandenabeele group is supported by Belgian grants (Interuniversity Attraction Poles, IAP 7/32), Flemish grants (Research Foundation Flanders: FWO G.0875.11, FWO G.0973.11, FWO G.0A45.12N, FWO G.0172.12, FWO G.0787.13N, FWO G.0607.13N, FWO KAN 31528711, FWO KAN 1504813N, FWO G0E04.16N), Methusalem grant (BOF09/01M00709 and BOF16/MET_V/007), Ghent University grants (MRP, GROUP-ID consortium, BOF14/GOA/019), a grant from the Foundation Against Cancer (2012-188) and grants from VIB. P.V. holds Methusalem grants (BOF09/01M00709 and BOF16/MET_V/007).

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Authors and Affiliations

  1. Molecular Signaling and Cell Death Unit, VIB Inflammation Research Center, Ghent, Belgium

    Sasker Grootjans, Behrouz Hassannia, Iris Delrue, Vera Goossens, Bartosz Wiernicki, Yves Dondelinger, Mathieu J M Bertrand, Dmitri V Krysko, Peter Vandenabeele & Tom Vanden Berghe

  2. Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium

    Sasker Grootjans, Behrouz Hassannia, Iris Delrue, Vera Goossens, Bartosz Wiernicki, Yves Dondelinger, Mathieu J M Bertrand, Dmitri V Krysko, Peter Vandenabeele & Tom Vanden Berghe

  3. Gnomixx, Ghent, Belgium

    Marnik Vuylsteke

  4. Methusalem Program, Ghent University, Ghent, Belgium

    Peter Vandenabeele

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  1. Sasker Grootjans
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Contributions

S.G. and Y.D. developed the protocol. S.G., M.V., V.G., T.V.B. and P.V. designed the research, interpreted the data and wrote the paper. S.G., B.H., I.D. and B.W. carried out the experiments. M.V. performed the repeated-measurements data analysis. M.J.M.B. and D.V.K. provided critical remarks and discussion.

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Correspondence to Sasker Grootjans or Tom Vanden Berghe.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Validation of the FAN assay

a, The linear range for SYTOX Green decreases at lower concentrations (< 5μM) even if signal intensity is below the saturation level of the detector. b- c, Cell membrane permeabilization kinetics (b) and caspase activation (c) in IMR-32 cells treated with 2 μM Staurosporin (STS). d- e The effect of DEVD-amc on propidium iodide (PI) intensity and vice versa. Fas-overexpressing L929 cells were treated with 250 ng/ml anti-Fas antibody in the presence or absence of 20 μM DEVD-amc or 10μM PI, and amc intensity (d) and PI intensity (e) was measured 30 h later. f, The effect of phenol red on PI intensity is minimal for permeabilized L929 cells. g- h, Cell membrane permeabilization kinetics measured with 10μM PI (g) and caspase activation (h) in L929-Fas cells treated with anti-Fas Ab (250 ng/ml). The same plate was measured at 6 and 30 hours after stimulation, and returned to the tissue culture incubator in the interval time. Error bars = standard error of the mean, obtained from three independent experiments (n=3), each measured in triplicate. ***, p ≤ 0.001. Non-significant differences are not indicated. AV, arbitrary values.

Supplementary Figure 2 Example of a plate lay-out to determine to optimal combination of gain setting, SYTOX Green concentration and cell number.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1 and 2, Supplementary Table and Supplementary Note (PDF 747 kb)

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Grootjans, S., Hassannia, B., Delrue, I. et al. A real-time fluorometric method for the simultaneous detection of cell death type and rate. Nat Protoc 11, 1444–1454 (2016). https://doi.org/10.1038/nprot.2016.085

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