YILIMART - Annexin V Micro Bead Kit

The Annexin V MicroBead Kit was developed for positive selection or depletion of cells based on their ability to bind Annexin V. The Annexin V MicroBead Kit includes Annexin V MicroBeads and Annexin V Binding Buffer.

Background information

In most normal, viable eukaryotic cells the negatively charged phospholipid phosphatidylserine (PS) is located in the cytosolic leaflet of the plasma membrane lipid bilayer

¹

. PS redistribution from the inner to the outer leaflet is an early and widespread event during apoptosis

^1,2

. However, in necrosis, PS becomes accessible due to the disruption of membrane integrity

²

. Apart from necrosis and apoptosis, PS also becomes accessible in activated platelets

³

, in certain cell anomalies such as sickle cell anaemia

⁴

, in erythrocyte senescence

⁵

, upon degranulation of mast cells

¹⁰

, and in certain stages of B cell differentiation

¹¹

. PS exposure also serves as a trigger for the recognition and removal of cells by macrophages

^6,9

.

Annexin V is a 35 kDa phospholipid-binding protein and a major cell membrane component of macrophages and other phagocytic cell types. Annexin V has a high affinity to PS in the presence of physiological concentrations of calcium (CA

²⁺

)

⁷

and has already been used to isolate cells with exposed PS using MACS

^®

MicroBeads.

^4,8

Applications

The Annexin V MicroBead Kit is used for the isolation of cells with exposed PS, including sickle cells and apoptotic cells. The Annexin V MicroBead Kit has been further applied to the purification of apoptotic and non-apoptotic neutrophils to study the intracellular signaling pathways that regulate neutrophil survival. In a study on the roles of different cytosolic phospholipase A2s in apoptosis, the Annexin V MicroBead Kit has been used for sensitive assessment of PS externalization.

Columns

For positive selection: MS, LS or XS Columns. For depletion: LD, CS or D Columns.

Figure 1

Enrichment of dead (apoptotic/necrotic) cells using the Annexin V MicroBead Kit. Murine thymocytes were treated with CD95 (anti-Fas) monoclonal antibody for 3 hours at 37 °C. PS-exposing cells were enriched by labeling with Annexin V MicroBeads followed by separation on an MS Column in the magnetic field of a MiniMACS™ Separator. Cell fractions were stained with Annexin V-FITC.

A:	B:
Murine thymocytes after treatment with CD95 (anti-Fas) antibody	Positive fraction enriched for PS-exposing cells

Figure 2
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Figure 2

Isolation of PS-exposing cells using the Annexin V MicroBead Kit.

Isolation of PS-exposing cells using the Annexin V MicroBead Kit.

Selected references
1. Koopman, G. et al. (1994) Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 84: 1415-1420
2. Martin, S. J. et al. (1995) Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J. Exp. Med. 182: 1545-1556
3. Thiagarajan, P. et al. (1990) Binding of annexin V/placental anticoagulant protein I to platelets. Evidence for phosphatidylserine exposure in the procoagulant response of activated platelets. J. Biol. Chem. 265: 17420-17423
4. Kuypers, F. A. et al. (1996) Detection of altered membrane phospholipid asymmetry in subpopulations of human red blood cells using fluorescently labeled annexin V. Blood 87: 1179-1187
5. Schroit, A. J. et al. (1991) Transbilayer movement of phospholipids in red cell and platelet membranes. Biochim. Biophys. Acta 1071: 313-329
6. Fadok, V. A. et al. (1992) Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J. Immunol. 148: 2207-2216
7. Moss, S. E. et al. (1991) Diversity in the Annexin family. In: Novel Calcium Binding Proteins, Springer Verlag : 535-566
8. Rickers, A. et al. (1998) Inhibition of CPP32 blocks surface IgM-mediated apoptosis and D4-GDI cleavage in human BL60 Burkitt lymphoma cells. Eur. J. Immunol. 28: 296-304
9. Fadok, V. A. et al. (2000) A receptor for phosphatidylserine-specific clearance of apoptotic cells. Nature 405: 85-90
10. Demo et al. (1999) Quantitative measurement of mast cell degranulation using a novel flow cytometric annexin-V binding assay. Cytometry 36: 340-348
11. Dillon, S. R. et al. (2001) Annexin V binds to positively selected B cells. J. Immunol. 166: 58-71
12. Dressel, R. et al. (2000) Heat shock protein 70 is able to prevent heat shock-induced resistance of target cells to CTL. J. Immunol. 164(5): 2362-2371
13. Khwaja, A. and Tatton, L. (1999) Caspase-mediated proteolysis and activation of protein kinase Cdelta plays a central role in neutrophil apoptosis. Blood 94: 291-301
14. Atsumi, G. et al. (2000)
  
  Distinct roles of two intracellular phospholipase A2s in fatty acid release in the cell death pathway. Proteolytic fragment of type IVA cytosolic phospholipase A2alpha inhibits stimulus-induced arachidonate release, whereas that of type VI Ca
  ²⁺
  -independent phospholipase A2 augments spontaneous fatty acid release.
  
  J. Biol. Chem. 275(24): 18248-18258

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Annexin V Micro Bead Kit

For research use only

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Background information

Applications

Columns

Figure 1

Figure 2

Figure 2

Selected references

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