Abstract
Cell death by apoptosis has been studied for many years using fluorescently labeled annexin V. Annexin V shows high affinity for the phosphatidylserine that becomes enriched in the outer leaflet of the plasma membrane during apoptosis, but not necrosis, allowing differentiation between the two types of cell death. In this chapter we detail two methods for the purification of annexin V. The first is an untagged recombinant protein using a three step Fast Protein Liquid Chromatography (FPLC) method, and the second using a single step purification protocol via a glutathione S-transferase (GST) tag. Labeling of the resulting annexin V with a fluorescent dye to allow visualization of the protein is also explained. Finally, two methods are described in which a fluorescently labeled derivative of annexin V is used to detect apoptosis, namely the in vitro method of fluorescence-activated cell sorting (FACS) where fluorescent annexin V is used to differentiate apoptotic and necrotic cells within a population; and detection of apoptosing retinal cells (DARC) allowing the identification of apoptotic cells in the retina in vivo.
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References
Fadok, V. A., Voelker, D. R., Campbell, P. A., Cohen, J. J., Bratton, D. L. and Henson, P. M. (1992a) Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages J Immunol 148, 2207–16.
Fadok, V. A., Savill, J. S., Haslett, C., Bratton, D. L., Doherty, D. E., Campbell, P. A. and Henson, P. M. (1992b) Different populations of macrophages use either the vitronectin receptor or the phosphatidylserine receptor to recognize and remove apoptotic cells J Immunol 149, 4029–35.
Andree, H. A., Reutelingspergerm, C. P., Hauptmann, R., Hemker, H. C., Hermens, W. T. and Willems, G. M. (1990) Binding of vascular anticoagulant alpha (VAC alpha) to planar phospholipid bilayers J Biol Chem 265, 4923–8.
Koopman, G., Reutelingsperger, C. P., Kuijten, G. A., Keehnen, R. M., Pals, S. T. and van Oers, M. H. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis Blood 84, 1415–20.
Cordeiro, M. F., Guo, L., Luong, V., Harding, G., Wang, W., Jones, H. E., Moss, S. E., Sillito, A. M. and Fitzke, F. W. (2004) Real-time imaging of single nerve cell apoptosis in retinal neurodegeneration Proc Natl Acad Sci USA 101, 13352–6.
Guo, L., Salt, T. E., Maass, A., Luong, V., Moss, S. E., Fitzke, F. W. and Cordeiro, M. F. (2006) Assessment of neuroprotective effects of glutamate modulation on glaucoma-related retinal ganglion cell apoptosis in vivo Invest Ophthalmol Vis Sci. 47, 626–33.
Guo, L., Salt, T. E., Luong, V., Wood, N., Cheung, W., Maass, A., Ferrari, G., Russo-Marie, F., Sillito, A. M., Cheetham, M. E., Moss, S. E., Fitzke, F. W. and Cordeiro MF. (2007) Targeting amyloid-beta in glaucoma treatment Proc Natl Acad Sci USA 104, 13444–9.
Hofstra, L., Liem, I. H., Dumont, E. A., Boersma, H. H., van Heerde, W. L., Doevendans, P. A., De Muinck, E., Wellens, H. J., Kemerink, G. J., Reutelingsperger, C. P. and Heidendal, G. A. (2000) Visualisation of cell death in vivo in patients with acute myocardial infarction Lancet 356, 209–12.
Thimister, P. W., Hofstra, L., Liem, I. H., Boersma, H. H., Kemerink, G., Reutelingsperger, C. P. and Heidendal, G. A. (2003) In vivo detection of cell death in the area at risk in acute myocardial infarction J Nucl Med 44, 391–6.
Belhocine, T., Steinmetz, N., Hustinx, R., Bartsch, P., Jerusalem, G., Seidel, L., Rigo, P. and Green, A. (2002) Increased uptake of the apoptosis-imaging agent (99 m) Tc recombinant human Annexin V in human tumors after one course of chemotherapy as a predictor of tumor response and patient prognosis Clin Cancer Res 8, 2766–74.
Kartachova, M. S., Valdés Olmos, R. A., Haas, R. L., Hoebers, F. J., van Herk, M. and Verheij, M. (2008) 99mTc-HYNIC-rh-annexin-V scintigraphy: visual and quantitative evaluation of early treatment-induced apoptosis to predict treatment outcome Nucl Med Commun 29, 39–44.
Kurihara, H., Yang, D. J., Cristofanilli, M., Erwin, W. D., Yu, D. F., Kohanim, S., Mendez, R. and Kim, E. E. (2008) Imaging and dosimetry of 99mTc EC annexin V: preliminary clinical study targeting apoptosis in breast tumors Appl Radiat Isot 66, 1175–82.
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Coxon, K.M., Duggan, J., Cordeiro, M.F., Moss, S.E. (2011). Purification of Annexin V and Its Use in the Detection of Apoptotic Cells. In: Cree, I. (eds) Cancer Cell Culture. Methods in Molecular Biology, vol 731. Humana Press. https://doi.org/10.1007/978-1-61779-080-5_24
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DOI: https://doi.org/10.1007/978-1-61779-080-5_24
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