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Monitoring the progression of cell death and the disassembly of dying cells by flow cytometry

Nature Protocols volume 11, pages 655–663 (2016)Cite this article

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Abstract

The use of annexin A5 (A5) and either propidium iodide or 7-aminoactinomycin D (PI/7-AAD) stains to measure cell death by flow cytometry has been considered the gold standard by most investigators. However, this widely used method often makes the assumption that there are only three types of particles in a sample: viable, apoptotic and necrotic cells. To study the progression of cell death in greater detail, in particular how apoptotic cells undergo fragmentation to generate membrane-bound vesicles known as apoptotic bodies, we established a flow cytometry–based protocol to accurately and rapidly measure the cell death process. This protocol uses a combination of A5 and TO-PRO-3 (a commercially available nucleic acid–binding dye that stains early apoptotic and necrotic cells differentially), and a logical seven-stage analytical approach to distinguish six types of particles in a sample, including apoptotic bodies and cells at three different stages of cell death. The protocol requires 1–5 h for sample preparation (including induction of cell death), 20 min for staining and 5 min for data analysis.

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Figure 1: Schematic of A5-FITC binding and TO-PRO-3 uptake by cells and cell fragments.
Figure 2: Flow cytometry plots of human Jurkat T cells for optimal data acquisition and analysis.
Figure 3: Electronic gating strategy for analyzing cell death and apoptotic cell disassembly.
Figure 4: Identification of cells at different stages of cell death and cell fragments based on different analytical approaches.

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Acknowledgements

This work was supported by grants from the National Health and Medical Research Council of Australia (nos. APP1013584 and APP1082383), La Trobe University (nos. RFA2014 and RFA2015) and a Ramaciotti Establishment Grant to I.K.H.P.

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  1. Lanzhou Jiang and Rochelle Tixeira: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia

    Lanzhou Jiang, Rochelle Tixeira, Sarah Caruso, Georgia K Atkin-Smith, Amy A Baxter, Stephanie Paone, Mark D Hulett & Ivan K H Poon

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Contributions

L.J., R.T. and I.K.H.P. designed, performed and analyzed most of the experiments with help and input from S.C., G.K.A.-S. and S.P. A.A.B., M.D.H., L.J. and I.K.H.P. designed and carried out experiments with rNaD1. I.K.H.P., L.J. and R.T. wrote the manuscript with input from co-authors.

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Correspondence to Ivan K H Poon.

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Integrated supplementary information

Supplementary Figure 1 Traditional electronic gating strategy for analysing cell death based on A5-FITC and 7-AAD.

a, Flow cytometry analysis showing the traditional two-stage electronic gating strategy used to identify viable cells, A5+ early apoptotic cells and necrotic cells. b, Flow cytometry analysis displaying each type of cells gated according to a has distinctive levels of A5-FITC and 7-AAD staining. Human Jurkat T cells were induced to undergo apoptosis by anti-Fas treatment (31.25 ng ml-1, 4 h). Samples were stained with A5-FITC (1 in 200 dilution), 7-AAD (1 μg ml-1) and TO-PRO-3 (0.5 μM) in 1x A5 Binding Buffer. 30,000 events (cells and cell fragments) were recorded by the FACSCanto II flow cytometer and resultant data analysed by the FlowJo 8.8.6 software. The TO-PRO-3 parameter was not used during data analysis.

Supplementary Figure 2 The nucleic acid-binding dye TO-PRO-3 stains viable, early apoptotic and necrotic cells differentially.

a, Histogram showing TO-PRO-3 uptake by viable, A5- early apoptotic, A5+ early apoptotic and necrotic cells. b, Uptake of TO-PRO-3 by cells at different stages of cell death. Data is presented as median fluorescence intensity (MFI) relative to viable cells (n = 3, experimental replicates). Human Jurkat T cells were induced to undergo apoptosis by anti-Fas treatment (31.25 ng ml−1, 4 h). Samples were stained with A5-FITC (1 in 200 dilution), 7-AAD (1 μg ml−1) and TO-PRO-3 (0.5 μM) in 1x A5 Binding Buffer. 30,000 events (cells and cell fragments) were recorded by the FACSCanto II flow cytometer. Resultant data were analysed by the FlowJo 8.8.6 software using the electronic gating strategy as described in Figure 3. Alternative STAGE 1 gating step was used to identify necrotic cells based on the 7-AAD parameter. Error bars represent standard error of the mean. Data are representative of at least two independent experiments.

Supplementary Figure 3 Representative images of typical particles in each stage of cell death.

ImageStream analysis of particles gated using the strategy as described in Poon et al., Nature 507, 329–334 (2014). Human Jurkat T cells were induced to undergo apoptosis by anti-Fas treatment (250 ng ml−1, 4 h). Samples were stained with A5-FITC (1 in 200 dilution), 7-AAD (1 μg ml−1) and TO-PRO-3 (0.5 μM) in 1x A5 Binding Buffer. Cells and cell fragments were recorded by the ImageStreamX Mark II and resultant data analysed by the IDEAS software. The data shown were previously published in Poon et al., Nature 507, 329–334 (2014).

Supplementary Figure 4 Electronic gating strategy for analysing cell death based only on TO-PRO-3.

Flow cytometry analysis showing a one-stage electronic gating strategy used to identify viable cells, early apoptotic cells (A5− and A5+), necrotic cells, as well as apoptotic bodies and debris. Human Jurkat T cells were induced to undergo apoptosis by anti-Fas treatment (31.25 ng ml−1, 4 h). Samples were stained with A5-FITC (1 in 200 dilution), 7-AAD (1 μg ml−1) and TO-PRO-3 (0.5 μM) in 1x A5 Binding Buffer. 30,000 events (cells and cell fragments) were recorded by the FACSCanto II flow cytometer and resultant data analysed by the FlowJo 8.8.6 software. The A5-FITC and 7-AAD parameter was not used during data analysis.

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Jiang, L., Tixeira, R., Caruso, S. et al. Monitoring the progression of cell death and the disassembly of dying cells by flow cytometry. Nat Protoc 11, 655–663 (2016). https://doi.org/10.1038/nprot.2016.028

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