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Annexin A5 (ANXA5) Protein

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Protein Name
Reactivity
40
15
10
7
3
3
3
3
1
1
Host
61
9
8
2
1
1
Application
Fluorescence Microscopy (FM), Flow Cytometry (FACS)
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Purpose A bright red color fluorescent reagent for detecting of the early stages of apoptosis.
Sample Type Cell Culture
Characteristics The Annexin V-Cy3 conjugate contains the bright red fluorescent probe that can be used for detection of apoptosis by fluorescence microscopy with a rhodamine filter or by flow cytometry. Cy3 yields red fluorescence with a lambdamax emission of 570 nm.
Background During apoptosis, phosphatidylserine (PS) is translocated from the cytoplasmic face of the plasma membrane to the cell surface. Annexin V has a strong, Ca2+-dependent affinity for PS and therefore serves as a probe for detecting apoptosis.
Research Area Cardiovascular, Atherosclerosis, Apoptosis/Necrosis, Coagulation
Pathways Apoptosis
Application Notes Detection method: Flow cytometry (Ex/EM = 543/570 nm) using FL2 channel and fluorescence microscopy
Protocol A. Incubation of cells with Annexin V-Cy3:
1. Induce apoptosis by desired methods.
2. Collect 1 x 10^5 cells by centrifugation.
3. Resuspend cells in 500 µL of 1X Annexin V Binding Buffer.
4. Add 1 µL of Annexin V-Cy3.
5. Incubate at room temperature for 5 min in the dark.
Proceed to B or C below depending on method of analysis.

B. Quantification by Flow Cytometry:
Analyze cells by flow cytometry (Ex = 543 nm, Em = 570 nm) using FL2 channel. For adherent cells, trypsinize and gently wash cells with serum-containing medium before incubation with Annexin V-Cy3 (A.3-5).

C. Detection by Fluorescence Microscopy:
1. Place the cell suspension from Step A.5 on a glass slide, and cover with a glass coverslip. For analyzing adherent cells, grow cells directly on a coverslip. Following incubation (A.5), invert coverslip on a glass slide and visualize cells. The cells can also be washed with 1X Annexin V Binding Buffer and fixed in 2 % formaldehyde before visualization. (Cells must be incubated with Annexin V-Cy3 before fixation because any cell membrane disruption can cause nonspecific binding of annexin V to PS on the inner surface of the cell membrane.)
2. Observe the cells under a fluorescence microscope using a rhodamine filter. Cells that have bound Annexin V-Cy3 will show bright red staining on the plasma membrane.
Restrictions For Research Use only
Handling Advice Do not freeze!
Storage 4 °C
Expiry Date 12 months
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Chen, Sandford, Nicholas: "Intracellular signaling mechanisms and activities of human herpesvirus 8 interleukin-6." in: Journal of virology, Vol. 83, Issue 2, pp. 722-33, 2008 (PubMed).

Sato, Sanjo, Tsujimura et al.: "TAK1 is indispensable for development of T cells and prevention of colitis by the generation of regulatory T cells." in: International immunology, Vol. 18, Issue 10, pp. 1405-11, 2006 (PubMed).

Zencak, Lingbeek, Kostic et al.: "Bmi1 loss produces an increase in astroglial cells and a decrease in neural stem cell population and proliferation." in: The Journal of neuroscience : the official journal of the Society for Neuroscience, Vol. 25, Issue 24, pp. 5774-83, 2005 (PubMed).

Ruiz-Vela, Opferman, Cheng et al.: "Proapoptotic BAX and BAK control multiple initiator caspases." in: EMBO reports, Vol. 6, Issue 4, pp. 379-85, 2005 (PubMed).

Breckenridge, Nguyen, Kuppig et al.: "The procaspase-8 isoform, procaspase-8L, recruited to the BAP31 complex at the endoplasmic reticulum." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 99, Issue 7, pp. 4331-6, 2002 (PubMed).