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Annexin V Binding Buffer

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Regulatory Status
RUO
Other Names
Annexin-V-Binding Buffer
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Product Citations
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422201 100 mL 36 CHF
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Description

Annexin V Binding Buffer has been formulated for flow cytometric labeling of apoptotic cells with Annexin V reagents.  The buffer is provided as a ready-to-use solution.

Product Details
Technical Data Sheet (pdf)

Product Details

Storage & Handling
Store between 2°C and 8°C.
Application

FC - Quality tested
Recommended Usage

Annexin V Binding Buffer is recommended for use with Annexin V reagents. Cells should be resuspended in Annexin V Binding Buffer at a concentration 1x106/ml and then combined with Annexin V reagents at the recommended volumes.

Application Notes

Annexin V Staining
1. Wash cells twice with cold BioLegend cell staining buffer (cat # 420201) and then resuspend cells in Annexin V Binding Buffer (cat # 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 fluorochrome conjugated Annexin V.
4. Add 10 µl of PI solution (cat # 421301) or 7-AAD (cat # 420403/420404).
5. Gently vortex the cells and incubate for 15 min at RT (25 °C) in the dark.
6. Add 400 µl of Annexin V Binding Buffer (cat # 422201) to each tube. Analyze by flow cytometry.


For a better experience detecting apoptosis, we now recommend Apotracker™. Cell staining with Apotracker™ is Calcium independent. Thus, no special buffers are required, and the protocol can be shortened for single-step co-staining with other reagents.

Application References

(PubMed link indicates BioLegend citation)
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Antigen Details

Gene ID
NA

Related FAQs

How is your Annexin made and what sequence does it cover?

It is made in E. coli, covering human aa Met1-Asp320.

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.

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.

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.

Can I freeze Annexin V conjugates?

It should not be frozen as it will lead to loss of biological activity due to dimerization.

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.

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