Jurkat cells were either untreated (left) or treated for 4 hours with 10µM camptothecin (right) and then stained with the Annexin V-APC Apoptosis Detection Kit and Propidium Iodide Staining Solution (PI) (cat. 00-6990). The Annexin V-positive, PI-negative population contains early stage apoptotic cells.
Contents: Annexin V-APC Apoptosis Detection Kit Catalog Number: 88-8007 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.
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Cat. No.
Size
Price
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88-8007-72
50 tests
88-8007-74
200 tests
Note: Several countries will continue to be supplied via distributors. Country specific prices may apply.
Flow Cytometry Product Notes: Test Sizes: To accommodate multicolor flow cytometry, eBioscience is in the process of reducing test size volumes from 20 µl to 5 µl. Please check your antibody vial for the recommended test size. Fluorochrome Replacements: eBioscience is in the process of replacing all Pacific Blue® and APC-Alexa Fluor® 750 conjugated products with eFluor™ 450 and APC-eFluor™ 780 conjugated products, respectively.
Description
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
10X Binding Buffer: Annexin V 10x Binding Buffer is a stock solution that must be diluted prior to use with distilled water. Dilute 1 part Binding Buffer with 9 parts distilled water.. -50 Test kit comes with 30 ml (Cat. No. 00-0055-43)
-200 Test kit comes with 100 ml (Cat. No. 00-0055-56) Annexin V-APC: -50 or 200 Test kit (5µl/test)
Applications Reported
For research use only, not for diagnostic or therapeutic use. Annexin V-APC Apoptosis Detection Kit has been reported for use in flow cytometric analysis.
Applications Tested
Annexin V-APC Apoptosis Detection Kit has been pre-titrated and tested on Jurkat cells treated with 10µM Camptothecin for 4 hours. This can be used at 5µl per million cells in a 100µl total staining volume.
Special Notes
This kit is compatible with eBioscience IC Fixation Buffer (cat. 00-8222) for intracellular staining. It is recommended that eBioscience Fixable Viability Dyes (cat. 65-0863, and 65-0865) are used for viability staining for all intracellular staining protocols.
References
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
Protocol
To view protocol, please download the TDS by clicking the PDF button above.
Legal
Pat. No. EP 181 465 B2, EP 0509 026, USP 5,066,787
