Abstract
The number of investigators using cell death analysis applications has greatly expanded since the introduction of flow cytometry. The Annexin V/propidium iodide (PI) method is among the most commonly used procedures and allows users to determine if cells are viable, apoptotic, or necrotic, based on changes in membrane lipid composition, integrity, and permeability. Unfortunately, PI can intercalate into RNA, in addition to DNA, which contributes to a large number of events showing PI staining within the cytoplasmic compartment. We show that this occurs across a broad range of animal primary cells and commonly used cell lines, and is most prevalent in large cells (nuclear:cytoplasmic ratio <0.5). Any cellular system where RNA levels change throughout an experiment will be particularly affected, such as those that utilize virally infected cells. As two examples, we highlight our recent work on cells infected with vesicular stomatitis virus (VSV), an RNA virus, and herpes simplex virus-1 (HSV-1), a DNA virus. Similarly, these issues are relevant to experimental systems where cells have increased RNA content such as during genotoxic stress, following exposure to cell cycle arrest drugs such as thymidine or hydroxyurea, or where developmental progression promotes discrete changes in cellular RNA synthesis. This chapter outlines a modified Annexin V/PI method that addresses cytoplasmic RNA staining issues to allow for accurate assessment of cell death. This protocol takes advantage of an additional cellular permeability caused by fixation to promote RNase A entry into the cell. Based on our observations, cell morphological parameters are well maintained and less than 5 % of total cellular events exhibit cytoplasmic PI staining under this protocol.
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References
Muppidi J, Porter M, Siegel RM (2004) Measurement of apoptosis and other forms of cell death. Curr Protoc Immunol Chapter 3, Unit 3. 17
Green DR (2011) Means to an end: apoptosis and other cell death mechanisms. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
Kroemer G, Dallaporta B, Resche-Rigon M (1998) The mitochondrial death/life regulator in apoptosis and necrosis. Annu Rev Physiol 60:619–642
Overbeeke R, Steffens-Nakken H, Vermes I, Reutelingsperger C, Haanen C (1998) Early features of apoptosis detected by four different flow cytometry assays. Apoptosis 3:115–121
Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C (1995) A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J Immunol Methods 184:39–51
Telford WG, Komoriya A, Packard BZ (2004) Multiparametric analysis of apoptosis by flow and image cytometry. Methods Mol Biol 263:141–160
Vermes I, Haanen C, Reutelingsperger C (2000) Flow cytometry of apoptotic cell death. J Immunol Methods 243:167–190
Darzynkiewicz Z, Bruno S, Del Bino G, Gorczyca W, Hotz MA, Lassota P, Traganos F (1992) Features of apoptotic cells measured by flow cytometry. Cytometry 13:795–808
Faleiro L, Lazebnik Y (2000) Caspases disrupt the nuclear-cytoplasmic barrier. J Cell Biol 151:951–959
Deitch AD, Law H, deVere WR (1982) A stable propidium iodide staining procedure for flow cytometry. J Histochem Cytochem 30:967–972
Fried J, Perez AG, Clarkson BD (1976) Flow cytofluorometric analysis of cell cycle distributions using propidium iodide. Properties of the method and mathematical analysis of the data. J Cell Biol 71:172–181
http://apps.isiknowledge.com/summary.do?qid=17&product=UA&SID= 3EdJcO897oK2Gfd6EH4&search_mode = Refine. (2009) Web of knowledge search
Zuba-Surma EK, Kucia M, Abdel-Latif A, Lillard JW Jr, Ratajczak MZ (2007) The ImageStream system: a key step to a new era in imaging. Folia Histochem Cytobiol 45:279–290
Henery S, George T, Hall B, Basiji D, Ortyn W, Morrissey P (2008) Quantitative image based apoptotic index measurement using multispectral imaging flow cytometry: a comparison with standard photometric methods. Apoptosis 13:1054–1063. doi:10.1007/s10495-008-0227-4
Rieger AM, Hall BE, Luong LT, Schang LM, Barreda DR (2010) Conventional apoptosis assays using propidium iodide generate a significant number of false positives that prevent accurate assessment of cell death. J Immunol Methods 358:81–92. doi:10.1016/j.jim.2010.03.019
Acknowledgments
This work was supported by Natural Sciences and Engineering Council of Canada (NSERC) research grant and an Alberta Agriculture Funding Consortium grant to D.R.B. A.M.R. is supported through an NSERC Vanier Canadian Graduate Scholarship and the University of Alberta (graduate teaching assistantship, Queen Elizabeth II graduate scholarship, and Dissertation Fellowship).
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Rieger, A.M., Barreda, D.R. (2016). Accurate Assessment of Cell Death by Imaging Flow Cytometry. In: Barteneva, N., Vorobjev, I. (eds) Imaging Flow Cytometry. Methods in Molecular Biology, vol 1389. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3302-0_15
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DOI: https://doi.org/10.1007/978-1-4939-3302-0_15
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