• Detection method- Flow cytometry (Ex = 543 nm; Em = 570 nm) and fluorescence microscopy
• Sample type- Living cells (suspension and adherant)
• Species reactivity- Mammalian
• Kit size- Convenient sizes (25, 100, 400 assays)
Features & Benefits:
• Simple one step staining procedure in 10 minutes
• Fast and convenient
• Cy3 shows brighter red fluorescence
• Annexin V-Cy3
• 1X Binding Buffer
Annexin V Apoptosis Detection Kit is based on the observation that soon after initiating apoptosis, cells translocate the membrane phosphatidyl-serine (PS) from the inner face of the plasma membrane to the cell surface. Once on the cell surface, PS can be easily detected by staining with a fluorescent conjugate of Annexin V, a protein that has a high affinity for PS. The one-step staining procedure takes only 10 minutes. Detection can be analyzed by flow cytometry or by fluorescence microscopy.
USAGE: For Research Use Only! Not For Use in Humans.
Annexin V-Cy3 Assay Protocol:A. Incubation of cells with Annexin V-Cy3
1. Induce apoptosis by desired method.
2. Collect 1-5 x 105
cells by centrifugation.
3. Resuspend cells in 500 µl of 1X Binding Buffer.
4. Add 5 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 Annexin V-Cy3 binding by flow cytometry (Ex = 543 nm; Em = 570 nm) using the phycoerythrin emission signal detector (usually FL2).
For analyzing adherent cells, gently trypsinize and wash cells once with serum-containing media 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. Cover the cells with a glass coverslip.
For analyzing adherent cells, grow cells directly on a coverslip. Following incubation (A.5), invert coverslip on glass slide and visualize cells. The cells can also be washed and fixed in 2% formaldehyde before visualization. (Cells must be incubated with Annexin V-Cy3 before fixation since 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 red staining in the plasma membrane.