Product Catalog

The analysis of phosphatidylserine on the outer leaflet of apoptotic cell membranes is performed using Annexin-V-Alexa 568 in combination with a DNA stain (e.g., BOBO-1) to differentiate apoptotic cells from necrotic cells. Apoptotic and necrotic cells can also be differentiated by cell surface markers.

• Detection flexibility: Analysis of results with flow cytometry and/or fluorescence microscopy
  Alexa is a red dye, which can be used for double or triple staining with other fluorescent markers (e.g., fluorescein-labeled surface markers)
• Accuracy: Accurately distinguish apoptotic cells from necrotic cells
• Stability of results: High photostability (long observation possible)
• Rapid and simple procedure

Alexa 568 is a fluorescent label. The Annexin-V-Alexa 568 conjugate can be easily analyzed on a 488-nm laser Fluorescent Analytical Cell Sorter (FACS) machine, or using a fluorescence microscope. Excitation is in the range of 488 - 596 nm with emission above 600 nm.
Additional secondary labeling is also possible. For example, membrane surfaces can be stained with fluorescein-labeled monoclonal antibodies for further cellular characterization.

In the early stages of apoptosis, changes occur on the cell surface. One of these plasma membrane alterations is the translocation of phosphatidylserine (PS) from the inner side of the plasma membrane to the outer layer, thereby exposing PS at the external surface of the cell. Fadok et al. showed that macrophages specifically recognize PS exposed on the surface of lymphocytes during apoptosis. This recognition and subsequent phagocytosis of apoptotic cells and bodies protects organisms from exposure to cellular compounds that lead to inflammation and usually accompany necrosis.
Annexin-V is a Ca²⁺-dependent, phospholipid-binding protein with a high affinity for PS. This protein can therefore be used as a sensitive probe for PS exposure on the outer leaflet of the cell membrane, and is suited to detect apoptotic cells. 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 (used for dye-exclusion tests [e.g., BOBO-1]) allows the discrimination of necrotic cells from the Annexin-V positively stained cell cluster.

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

Figure 1: Discrimination between apoptotic and necrotic U937 cells treated with camptothecin (CAM) and stained with  Annexin-V-Alexa 568 (red) and BOBO-1 (green).