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Annexin A5 (ANXA5) Protein

Details for Product No. ABIN412010, Supplier: Log in to see
Protein Name
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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