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Annexin V (or Annexin A5) is a member of the annexin family of intracellular proteins that binds to phosphatidylserine (PS) in a calcium-dependent manner. PS is normally only found on the intracellular leaflet of the plasma membrane in healthy cells, but during early apoptosis, membrane asymmetry is lost and PS translocates to the external leaflet. Fluorochrome-labeled Annexin V can then be used to specifically target and identify apoptotic cells. Annexin V Binding Buffer (Cat. No. 422201) is recommended for use with Annexin V staining. Annexin V binding alone cannot differentiate between apoptotic and necrotic cells. Therefore, we recommend using our Helix NP™ Blue (Cat. No. 425305), Helix NP™ Green (Cat. No. 425303) or Helix NP™ NIR (Cat. No. 425301). Early apoptotic cells will exclude 7-AAD and PI, while late stage apoptotic cells and necrotic cells will stain positively, due to the passage of these dyes into the nucleus where they bind to DNA.Product Details
- All mammalian species
- Phosphate-buffered solution, pH 7.2, containing 0.09% sodium azide and 0.2% (w/v) BSA (origin USA).
- The antibody was purified by affinity chromatography and conjugated with APC/Fire™ 750 under optimal conditions. The solution is free of unconjugated APC/Fire™ 750 and unconjugated antibody.
- Lot-specific (please contact technical support for concentration and total µg amount, or use our Lookup tool if you have a lot number.)
- Storage & Handling
- The antibody solution should be stored undiluted between 2°C and 8°C, and protected from prolonged exposure to light. Do not freeze.
FC - Quality tested
- Recommended Usage
Each lot of this product is quality control tested by immunofluorescent staining with flow cytometric analysis. For flow cytometric application, the suggested use of this reagent is 5 - 15 µg per 100,000 - million cells in a 100 µl volume of Annexin V Binding Buffer (Cat No. 422201). It is recommended that the reagent be titrated for optimal performance for each application.
* APC/Fire™ 750 has a maximum excitation of 650 nm and a maximum emission of 787 nm.
- Excitation Laser
Red Laser (633 nm)
- Application Notes
Annexin V Staining
1. Wash cells twice with cold BioLegend Cell Staining Buffer (Cat. No. 420201) and then resuspend cells in Annexin V Binding Buffer (Cat. No. 422201) at a concentration of 1x106 cells/ml.
2. Transfer 100 µl of cell suspension in 5 ml test tube.
3. Add 5 µl of APC/Fire™ 750 Annexin V.
4. Add 10 µl of PI solution (Cat. No. 421301) or 7-AAD (Cat. No. 420403/420404).
5. Gently vortex the cells, and incubate for 15 min at room temperature (25°C), in the dark.
6. Add 400 µl of Annexin V Binding Buffer (Cat. No. 422201) to each tube. Analyze by flow cytometry.
(PubMed link indicates BioLegend citation)
- Can I freeze Annexin V conjugates?
It should not be frozen as it will lead to loss of biological activity due to dimerization.
- Can I use RPMI during Annexin V staining?
It is best to follow protocol as described on the product data sheet. Moreover, RPMI 1640 has a relatively high concentration of phosphate and low calcium ion concentration, which negatively impacts Annexin binding to its target phosphatidylserine (PS). Measurement of cell death by using Annexin V may also be significantly affected by time of incubation on ice, calcium concentration, and type of medium.
- How does pH and staining temperature affect Annexin V-Phosphatidylserine binding?
Annexin-Phosphatidylserine binding is lost below pH 5.2 and with prolonged incubation over a temperature of 42°C.
- How is your Annexin made and what sequence does it cover?
It is made in E. coli, covering human aa Met1-Asp320.
- Is Annexin V suitable for conjugation with the Maxpar® kit for CyTOF®?
Maxpar® Labeling kits require the protein to be partially reduced, so the metal chelate can be introduced through an SH group in the hinge region of the reduced antibody. Human Annexin V contains only one Cysteine which was reported to be chemically inactive. Thus, the Maxpar® labeling protocol would not work with Annexin V, unless a free –SH group can be introduced to Annexin V. For more information regarding SH-mediated conjugation of Annexin V please consult published papers such as this one.
- Why do I need to use Annexin V Binding Buffer?
Annexin V binding requires the presence of calcium in the solution. So, we provide Annexin V Binding Buffer (cat # 422201), which is optimized for the best performance of Annexin V staining.
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