Introduction to the Annexin V Apoptosis Detection Kits

Phosphatidylserine is a glycerophospholipid, i.e. a glycerol backbone with two fatty acids attached in ester linkage to the first and second carbon and a serine attached through a phosphodiester linkage to the third carbon of the glycerol. Phosphatidylserines are actively held facing the cytosolic side of the cell membrane. However, when a cell undergoes apoptosis, phosphatidylserine is no longer restricted to the cytosolic side (1). When the phosphatidylserines flip to the extracellular surface of the cell, they act as a signal for macrophages to engulf the cells (2).

Annexin V (or Annexin A5) is a member of a calcium and phospholipid binding family of proteins with vascular anticoagulant activity. Although its function remains elusive, results from in vitro experiments indicate that it may play a role in the inhibition of blood coagulation by competing for phosphatidylserine (PS) binding sites with prothrombin (3,4). Its selective binding capacity to PS makes it an excellent probe for early apoptosis detection when coupled to a fluorophore like Fluorescein, or its derivative Fluorescein IsoThioCyanate (FITC) (5,6). ICT’s Annexin V kits come with either of these two green emitting fluorophores with the excitation/emission characteristics of 494/521 nm and 488/530 nm, respectively.

• Detect apoptosis and membrane permability with the Annexin V-FITC Apoptosis Assay Kit. This in vitro apoptosis assay employs the green fluorescent Annexin V-FITC reagent to label exposed phosphatidylserine in cultured cell samples. Detect membrane compromised cells, a trademark of late apoptosis or cell necrosis, with the live/dead stain, propidium iodide. Analyze the fluorescent signals by flow cytometry.
  
  
• ICT's Bovine Annexin V-Fluorescein, Canine Annexin V-Fluorescein, Chicken Annexin V-Fluorescein, Equine Annexin V-Fluorescein, and Swine Annexin V-Fluorescein Apoptosis Assay Kits provide a proven method for quickly and easily distinguishing two populations of dying cells from viable cells using recombinant fluorescein-conjugated bovine, canine, chicken, equine and swine annexin v-fluorescein (green fluorescence) and Propidium Iodide (red fluorescence). Results can be analyzed via flow cytometer.
  
  Kits based on Annexin V detection to measure apoptosis are very popular and generate many publications. For example, recent journal articles involving Annexin V include investigations on anti-inflammatory agents on apoptotic macrophages (7), on the search for a new generation of anti-cancer reagents (8), on the downregulation of microRNA in cancer (9), on metastasis (10) and on the ER stress response (11).

References

1. Fadok, V. A. et al. Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J Immunol 148, 2207-2216 (1992).
2. Verhoven, B., Schlegel, R. A. & Williamson, P. Mechanisms of phosphatidylserine exposure, a phagocyte recognition signal, on apoptotic T lymphocytes. J Exp Med 182, 1597-1601 (1995).
3. Andree, H. A. et al. Binding of vascular anticoagulant alpha (VAC alpha) to planar phospholipid bilayers. J Biol Chem 265, 4923-4928 (1990).
4. van Heerde, W. L., Poort, S., van ‘t Veer, C., Reutelingsperger, C. P. & de Groot, P. G. Binding of recombinant annexin V to endothelial cells: effect of annexin V binding on endothelial-cell-mediated thrombin formation. Biochem J 302 ( Pt 1), 305-312 (1994).
5. Koopman, G. et al. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 84, 1415-1420 (1994).
6. Vermes, I., Haanen, C., Steffens-Nakken, H. & Reutelingsperger, C. 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 (1995).
7. Mazur-Bialy, A. I., Pochec, E. & Zarawski, M. Anti-Inflammatory Properties of Irisin, Mediator of Physical Activity, Are Connected with TLR4/MyD88 Signaling Pathway Activation. Int J Mol Sci 18, doi:10.3390/ijms18040701 (2017).
8. Kasumbwe, K., Venugopala, K. N., Mohanlall, V. & Odhav, B. Synthetic Mono/di-halogenated Coumarin Derivatives and Their Anticancer Properties. Anticancer Agents Med Chem 17, 276-285 (2017).
9. Fan, J. Y. et al. miR-429 is involved in regulation of NF-kappaBactivity by targeting IKKbeta and suppresses oncogenic activity in cervical cancer cells. FEBS Lett 591, 118-128, doi:10.1002/1873-3468.12502 (2017).
10. Hu, X. et al. CCDC178 promotes hepatocellular carcinoma metastasis through modulation of anoikis. Oncogene 36, 4047-4059, doi:10.1038/onc.2017.10 (2017).
11. Dai, F. et al. BAP1 inhibits the ER stress gene regulatory network and modulates metabolic stress response. Proc Natl Acad Sci U S A 114, 3192-3197, doi:10.1073/pnas.1619588114 (2017).