The name Annexin pools over 160 different proteins. They are united by similar characteristics such as the ability to bind phosphatidylserine (PS) in a calcium dependent manner and a 70 amino acid repeat sequence called Annexin repeat.
While different types of Annexins can function as membrane scaffolds, annexin A-V is the most abundant membrane-bound Annexin scaffold. Annexin A-V can form 2-dimensional networks when bound to the phosphatidylserine unit of the membrane. Annexin A-V is effective in stabilizing changes in cell shape during endocytosis and exocytosis, as well as other cell membrane processes.
In normal viable cells, phosphatidylserine is located on the cytoplasmic surface of the cell membrane. However soon after initiating apoptosis, cells translocate the membrane phosphatidylserine from the inner face of the plasma membrane to the cell surface. Once on the cell surface, phosphatidylserine can be easily detected by staining with a fluorescent conjugate of Annexin V, which has a high affinity for phosphatidylserine. Therefore fluorochrome-labeled Annexin V is a reliable method to specifically target and identify apoptotic cells. The Annexin A5 affinity assay typically uses a conjugate of Annexin V and a fluorescent (e.g. FITC) or enzymatic label, biotin or other tags, or a radioelement, in a suitable buffer (Annexin V binding to PS is Ca2+ dependent). Detection can be analyzed by flow cytometry or by fluorescence microscopy. antibodies-online provides you Annexin V Apoptosis Kits from various suppliers.
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Annexin V binding alone cannot differentiate between apoptotic and necrotic cells. To help distinguish between the necrotic and apoptotic cells we offer different solutions.
One possibility is the addition of Propidium Iodide Solution (PI) (ABIN2669905) or 7-amino-actinomycin D (7-AAD) (ABIN2669212) . Early apoptotic cells will exclude both PI and 7-AAD, while late stage apoptotic cells and necrotic cells will stain positively, due to the passage of these dyes into the nucleus where they bind to DNA. When excited by 488 laser light, PI as well as 7-AAD fluorescence is detected in the far red range of the spectrum (650 nm long-pass filter).
SYTOX green dye (ABIN411998) is another possible addition, the assay can be directly performed on live cells, without the need of fixation. The SYTOX green dye is impermeant to live cells and apoptotic cells, but stains necrotic cells with intense green fluorescence by binding to cellular nucleic acids. After staining a cell population with Annexin V-FITC and SYTOX Green, apoptotic cells show green fluorescence, dead cells show a higher level of green fluorescence and live cells show little or no fluorescence.
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References and citations of our Annexin V Apoptosis Kits:
- Ramírez-Labrada, López-Royuela, Jarauta, Galán-Malo, Azaceta, Palomera, Pardo, Anel, Marzo, Naval: "Two death pathways induced by sorafenib in myeloma cells: Puma-mediated apoptosis and necroptosis." in: Clinical & translational oncology : official publication of the Federation of Spanish Oncology Societies and of the National Cancer Institute of Mexico, Vol. 17, Issue 2, pp. 121-32, 2015.
- Clarke, Weist, Walsh, Tenner: "Complement protein C1q bound to apoptotic cells suppresses human macrophage and dendritic cell-mediated Th17 and Th1 T cell subset proliferation." in: Journal of leukocyte biology, Vol. 97, Issue 1, pp. 147-60, 2015.
- Fu, Lv, Hua, He, Dong, Lele, Li, Zhai, Davis, Wang: "YAP regulates cell proliferation, migration, and steroidogenesis in adult granulosa cell tumors." in: Endocrine-related cancer, Vol. 21, Issue 2, pp. 297-310, 2014.
- Weng, Huang, Dong, Zhao, Zhou, Qu: "Inhibition of miR-17 and miR-20a by oridonin triggers apoptosis and reverses chemoresistance by derepressing BIM-S." in: Cancer research, Vol. 74, Issue 16, pp. 4409-19, 2014.