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Annexin A5 (ANXA5) Protein

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Protein Name
Reactivity
39
15
10
7
3
3
3
3
1
1
Host
52
9
9
7
2
1
1
Application
Fluorescence Microscopy (FM), Flow Cytometry (FACS)
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Purpose Annexin V-Cy5 is a bright fluorescent reagent for detecting of the early stages of apoptosis.
Sample Type Cell Culture
Characteristics Cy5 fluorescent dye produces an intense signal in the far-red region of the spectrum and therefore it is very useful for multiple labeling of cells with green and red colored fluorescent probes. Cy5 yields fluorescence with a lambdamax emission of 670 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 = 649 /670 nm) using Helium-Neon Laser and fluorescence microscopy
Protocol A. Incubation of cells with Annexin V-Cy5:
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-Cy5.
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 = 649 nm, Em = 670 nm) using Helium-Neon Laser. For adherent cells, trypsinize and gently wash cells with serum-containing medium before incubation with Annexin V-Cy5 (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 % formaldehyebefore visualization. (Cells must be incubated with Annexin V-Cy5 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 Cy5 filter or a FITC/Cy3/Cy5 triple band filter set (Chroma Technology) if you perform triple labeling with these dyes, or detect cells using CCD camera. Cells that have bound Annexin V-Cy5 will show bright red staining on the plasma membrane.
Restrictions For Research Use only
Storage 4 °C
Expiry Date 12 months
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