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Contents: Annexin V-FITC Apoptosis Detection Kit Catalog Number: 11-8005 Formulation: 50mM Tris, pH 7.4, 100mM NaCl, 1% BSA, 0.02% Sodium Azide Storage Conditions: Store at 4°C. DO NOT FREEZE. LIGHT-SENSITIVE MATERIAL.

Staining of Jurkat cells either untreated (left) or treated for 4 hours with 10µM camptothecin (right) with FITC Recombinant Human Annexin V and Propidium Iodide Staining Solution (PI) (cat. 00-6990). The Annexin V-positive, PI-negative population contains early stage apoptotic cells.

Available Formats of This Product
Cat. No.	Format	Excite (nm)	Emit (nm)	Reported Applications
88-8005	Annexin V-FITC Apoptosis Detection Kit	N/A	N/A	FC

Questions? Please consult our answers to frequently asked questions at http://www.ebioscience.com/faq.

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Description

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Annexins are a family of calcium-dependent phospholipid-binding proteins abundant in the eukaryotic kingdom. Annexin V's preferential binding partner is phosphatidylserine (PS). Under normal physiologic conditions, PS is predominantly located in the inner leaflet or cytosol-facing part of the plasma membrane. Upon initiation of apoptosis, PS loses its asymmetric distribution in the phospholipid bilayer and is translocated to the extracellular membrane leaflet where it identifies cells as targets of phagocytosis. Once on the outer surface of the membrane, PS can be detected by a fluorescently labeled annexin V in a Ca2+ -dependent manner.

In early stage apoptosis, the plasma membrane excludes viability dyes such as propidium iodide and 7-AAD, so these cells will only stain with Annexin V and serves as an early marker of apoptosis. However, in late stage apoptosis, the cell membrane loses integrity allowing Annexin V to access PS in the interior of the cell, as well. A viability dye such as 7-AAD or propidium iodide (PI) can be used to resolve these late stage apoptotic and necrotic cells (Annexin V and 7-AAD or PI double positive) from the early stage apoptotic cells (Annexin V positive, 7-AAD or PI negative).

Not included:
Propidium Iodide Staining Solution (cat. 00-6990)

Components:

1. 10X Binding Buffer: 1 ml. This is a 10X stock solution that must be diluted prior to use with distilled water. Dilute 1 part Binding Buffer with 9 parts distilled water.
2. Annexin V-FITC: 5μl/test

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Applications Reported

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For research use only, not for diagnostic or therapeutic use. FITC Recombinant Human Annexin V (Anxa5) has been reported for use in flow cytometric analysis.

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Applications Tested

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FITC Recombinant Human Annexin V (Anxa5) has been pre-titrated and tested on Jurkat cells treated with 10µM Camptothecin for 4 hours. This can be used at 5 μl per 1-5x10⁵ cells in a 100 μl total staining volume.

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References

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Andree HA, Reutelingsperger CP, Hauptmann R, Hemker HC, Hermens WT, Willems GM. Binding of vascular anticoagulant alpha (VAC alpha) to planar phospholipid bilayers. J Biol Chem. 1990; 265(9):4923-4928

Koopman G, Reutelingsperger CP, Kuijten GA, Keehnen RM, Pals ST, van Oers MH. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood. 1994; 84(5):1415-1420

Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J Immunol Methods. 1995; 184(1):39-51

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Related Products

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Cat. 00-6990    Propidium Iodide Staining Solution
Cat. 00-6993    7-AAD Viability Staining Solution
Cat. 88-8007    Annexin V-APC Apoptosis Detection Kit

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Annexin V Staining Protocol

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1. Dilute 10X binding buffer 1:10 in distilled water (1ml 10X Binding Buffer + 9ml dH₂0).
2. Wash cells once in PBS, then once in diluted Binding Buffer.
3. Resuspend cells in diluted Binding Buffer at 1-5x10⁶/ml.
4. Mix 100μl of the cell suspension and 5μl of fluorochrome-conjugated Annexin V.
5. Incubate 10-15 minutes at room temperature.
6. Wash cells in diluted Binding Buffer and resuspend in 200μl of diluted Binding Buffer.
7. Add 5μl of Propidium Iodide Staining Solution (cat. 00-6990).
8. Analyze by flow cytometry using the PE channel for Propidium Iodide.

Note: Due to the calcium dependence of the Annexin V : PS interaction, it is critical to avoid buffers containing EDTA or other calcium chelators during Annexin V experiments. Annexin V can only be used as a marker of apoptosis in cells where the plasma membrane is intact because destroying the integrity of the plasma membrane will allow non-specific binding of Annexin V to PS inside the cell.

Pat. No. EP 181 465 B2, EP 0509 026, USP 5,066,787