• Detection method- Flow cytometry (Ex = 488 nm; Em = 670 nm) and fluorescence microscopy
• Sample type- Live cells
• Species reactivity- Mammalian
• Kit size- Convenient sizes (25, 100, 400 assays)
• Applications- Detect early/middle stages of apoptosis; differentiate apoptosis from necrosis.
Features & Benefits:
• Simple one-step procedure; takes only 10 minutes
• Fast and convenient, no need of fixation of cells
• Annexin V-PE-Cy5
• 1X Binding Buffer
The Annexin V-PE-Cy5 Apoptosis Detection Kit is based on the observation that soon after initiating apoptosis, most cell types translocate the membrane phospholipid phosphatidylserine (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 strong natural affinity for PS. The one-step staining procedure takes only 10 minutes. In addition, the assay can be directly performed on live cells, without the need for fixation. Results can be analyzed by flow cytometry or fluorescence microscopy.
USAGE: For Research Use Only! Not For Use in Humans.
Annexin V-PE-Cy5 Assay Protocol:A. Incubation of cells with Annexin V-PE-Cy5
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-PE-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 Annexin V-PE-Cy5 binding by flow cytometry (Ex = 488 nm; Em = 670 nm).
For analyzing adherent cells, gently trypsinize and wash cells once with serum-containing media before incubation with Annexin V-PE-Cy5 (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-PE-Cy5 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 the PE-Cy5 filter.
Cells which have bound Annexin V-PE-Cy5 will show bright purple staining in the plasma membrane.