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The analysis of phosphatidylserine on the outer leaflet of apoptotic cell membranes is performed using Annexin-V-FLUOS. Additionally, labeling with propidium iodide (PI) or with a cell surface marker can be used for differentiation from necrotic cells.

Detection: Annexin-V-FLUOS can be directly detected in FACS analysis and immunochemistry without a secondary detection system.
Sample material: Cell lines and freshly isolated cells.
Specificity: Annexin-V binds in a calcium-dependent manner to negatively charged phospholipid surfaces, and shows high affinity for phosphatidylserine.

In the early stages of apoptosis, changes occur on the cell surface (e.g., translocation of phosphatidylserine [PS] from the interior side of the plasma membrane to the outer leaflet). This secretion of phosphatidylserine is the ideal basis for Annexin-V, which binds to PS with high affinity. Annexin-V is a Ca²⁺-dependent phospholipid-binding protein with a high affinity for PS. Since necrotic cells also expose PS as a result of lost membrane integrity, apoptotic cells must be differentiated from these necrotic cells. The simultaneous application of a DNA stain, which is used for dye-exclusion testing, allows the discrimination of necrotic cells from the Annexin-V positively stained cell cluster.

Contents
500 µl, ready-to-use solution, for 250 tests

1. Induce apoptosis.
2. Wash the cells in PBS.
3. Incubate cells with Annexin-V-FLUOS in a Hepes buffer that contains PI or a similar labeling reagent for cell surfaces.
4. Analyze samples under a fluorescence microscope or on a flow cytometer.

Figure 1: Discrimination between apoptotic and necrotic U937 cells treated with camptothecin. Late-stage apoptosis detected with Annexin-V-FLUOS (green), and counterstained with propidium iodide (necrotic cells).

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