- Regulatory Status
- RUO
- Other Names
- Annexin-V-Binding Buffer
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- Product Citations
- publications
Cat # | Size | Price | Quantity Check Availability | Save | ||
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422201 | 100 mL | 29€ |
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 DetailsProduct Details
- Storage & Handling
- Store between 2°C and 8°C.
- Application
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FC - Quality tested
- Recommended Usage
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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
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Annexin V Staining
- 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.
- Transfer 100 µL of cell suspension in 5 mL test tube.
- Add 5 µL of fluorochrome conjugated Annexin V.
- Stain with a viability dye, such as PI (Cat. No. 421301), 7-AAD (Cat. Nos. 420403 & 420404), or Helix NP dyes (Cat. Nos. 425301, 425303, & 425305), if desired.
- Gently vortex the cells and incubate for 15 min at RT (25°C) in the dark.
- Add 400* µL of Annexin V Binding Buffer (Cat. No. 422201) to each tube. *For more concentrated samples, add a minimum of 200 µl of Annexin V Binding Buffer in this step.
- 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.
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Application References
(PubMed link indicates BioLegend citation) -
- Vermes I, et al. 1995. J. Immunol. Methods 184:39.
- Soon PS, et al. 2013. Endocr Relat Cancer. 20:1. PubMed
- Turner JE, et al. 2013. J Exp Med. PubMed
- Lindner JM, et al. 2013. Mol Cell Biol. 33:4628. PubMed
- Turner JE, et al. 2013 J. Exp Med. 210:2951. PubMed
- Beggs KM, et al. 2014. Toxicol Sci. 137:91. PubMed
- Miyazawa M, et al. 2014. Mol Biol Cell. 25:2116. PubMed
- Burbulla LF, et al. 2014. Cell Death Dis. 5:1180. PubMed
- de Vires M, 2014. Am J Physiol Lung Cell Mol Physiol. 307:240. PubMed
- Sahin E, 2014. J. Immunol. 193:1717. PubMed
- Xiong R, et al. 2014. Toxicol Appl Pharmacol. 280:285. PubMed
- Thakran S, 2015. Invest Ophthalmol Vis Sci. 56:177. PubMed
- Product Citations
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Antigen Details
- Gene ID
- NA
Related FAQs
- How is your Annexin made and what sequence does it cover?
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It is made in E. coli, covering human aa Met1-Asp320.
- How does pH and staining temperature affect Annexin V-Phosphatidylserine binding?
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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?
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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?
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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?
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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®?
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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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