BD Pharmingen™ Annexin V-FITC Fluorescence Microscopy Kit
(RUO)
Description
The loss of plasma asymmetry is a key feature of apoptosis. In normal cells, membrane phospholipids are distributed asymmetrically between the inner and outer leaflets of the plasma membrane. For example, phosphatidylserine (PS), an aminophospholipid, is normally present in the inner leaflet of the plasma membrane. Early in apoptosis, before the loss of membrane integrity, PS translocates from the inner to the outer leaflet of the plasma membrane, exposing it to the external cellular environment at the surface of the plasma membrane. Annexin V is a 35-36 kDa, calcium-dependent, phospholipid-binding protein with a high affinity for PS and is used to indirectly monitor PS translocation. The Annexin V assay is perhaps the most widely used assay today, facilitated by the observation that PS translocation appears to be a universal apoptotic phenomenon. It has been detected in mammalian, insect, and plant cells under the action of most, if not all, triggers of apoptosis.
Preparation And Storage
The Annexin V was conjugated with FITC under optimum conditions, and unreacted FITC was removed.For a working solution (1X), dilute 1 part either 10X Annexin V Binding Buffer or 10X PBS Buffer to 9 parts distilled H20.
Recommended Assay Procedures
Annexin V-FITC binds to negatively charged phospholipid surfaces (Kd of ~5x10^-2) with a higher specificity for PS than most other phospholipids. Defined calcium and salt concentrations are required for Annexin V-FITC binding.
Annexin V-FITC Microscopy Staining Protocol
1. Wash cells twice with 1X PBS.
2. Wash cells once with 1X Annexin V Binding Buffer.
3. Stain cells with Annexin V-FITC diluted 1:10 in 1X Annexin V Binding Buffer for 15 min at RT. We typically use 2 ml total volume per Labtek II chamber slide (i.e., 200 µl Annexin V-FITC and 1.8 ml 1X Binding Buffer. For example, for a 4 well chambered slide, use 500 µl Annexin V-FITC solution/well.
4. Wash cells once with 1X Binding Buffer.
5. Add additional Binding Buffer and observe under the microscope. The amount of Binding Buffer added should be sufficient to ensure that the slide does not dry out during microscopic observation.
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
| Description | Quantity/Size | Part Number | EntrezGene ID |
|---|---|---|---|
| Annexin V-FITC | N/A | 51-8074KC | N/A |
| 10X Annexin V Binding Buffer | 50 mL (1 ea) | 51-66121E | N/A |
| 10X PBS Buffer | N/A | 51-6635KC | N/A |
Development References (2)
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Bossy-Wetzel E and Green D. Detection of apoptosis by Annexin V labeling. 2000; 322:15-18. (Clone-specific).
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Van Engeland, Nieland LJW, Ramaekers FCS, Schutte B, and Reutelingsperger CPM. . Annexin V-affinity assay: A review on an apoptosis detection system based on phosphatidylserine exposure. Cytometry. 1998; 31:1-9. (Biology).
Please refer to Support Documents for Quality Certificates
Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described
Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
