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PE Annexin V Apoptosis Detection Kit with 7-AAD

Pricing & Availability
Regulatory Status
RUO
Other Names
Annexin A5 Apoptosis Detection Kit
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Product Citations
publications
Annexin-V_PE_Antibody_FC_1_050714.jpg
Human T-cell leukemia cell line, Jurkat, treated (top) or non-treated (bottom) with LEAF™ purified anti-human CD95 (clone EOS9.1) for 4 hours, then stained with PE Annexin V Apoptosis Detection Kit with 7-AAD.
  • Annexin-V_PE_Antibody_FC_1_050714.jpg
    Human T-cell leukemia cell line, Jurkat, treated (top) or non-treated (bottom) with LEAF™ purified anti-human CD95 (clone EOS9.1) for 4 hours, then stained with PE Annexin V Apoptosis Detection Kit with 7-AAD.
  • Annexin-V_PE_Antibody_FC_2_050714.jpg
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Description

BioLegend's PE Annexin V Apoptosis Detection Kit with 7-AAD has been specifically designed for the identification of apoptotic and necrotic cells.

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 is recommended for use with Annexin V staining. Annexin V binding alone cannot differentiate between apoptotic and necrotic cells. To help distinguish between the necrotic and apoptotic cells we recommend use of our 7-amino-actinomycin D (7-AAD) solution. Early apoptotic cells will exclude 7-AAD, while late stage apoptotic cells will stain positively, due to the passage of these dyes into the nucleus where they bind to DNA.

7-AAD (7-amino-actinomycin D) has a high DNA binding constant and is efficiently excluded by intact cells. It is useful for DNA analysis and dead cell discrimination during flow cytometric analysis. When excited by 488 laser light, 7-AAD fluorescence is detected in the far red range of the spectrum (650 nm long-pass filter).

Product Details
Technical Data Sheet (pdf)

Product Details

Verified Reactivity
Human, Mouse, Rat
Reported Reactivity
Other Species
Concentration
Lot-specific (to obtain lot-specific concentration and expiration, please enter the lot number in our Certificate of Analysis online tool.)
Storage & Handling
Store between 2°C and 8°C. Do not freeze.

Caution: 7-AAD is a potential carcinogen. It is recommended that the user wear protective clothing, gloves, and eye/face protection in order to avoid contact with skin and eyes.
Application

FC - Quality tested
Recommended Usage

Staining Procedure:
1. Wash cells twice with cold BioLegend's Cell Staining Buffer, and then resuspend cells in Annexin V Binding Buffer at a concentration of 0.25-1.0 x 107 cells/mL.
2. Transfer 100 µL of cell suspension in a 5 mL test tube.
3. Add 5 µL of PE Annexin V.
4. Add 5 µL of 7-AAD Viability Staining Solution.
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 to each tube. Analyze by flow cytometry with proper machine settings.

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 fluorochrome conjugated Annexin V.
  4. 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.
  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. No. 422201) to each tube. *For more concentrated samples, add a minimum of 200 µl of Annexin V Binding Buffer in this step.
  7. 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)
  1. Maciel E, et al. 2014. Arch Biochem Biophys. 548:38. PubMed
Product Citations
  1. Pelletier A, et al. 2023. EMBO Rep. 24:e56156. PubMed
  2. Manolakou T, et al. 2023. J Cell Mol Med. 27:1192. PubMed
  3. Estep BK, et al. 2022. iScience. 25:105544. PubMed
  4. Ma Y, et al. 2022. Am J Physiol Heart Circ Physiol. 322:H622. PubMed
  5. Yu Y, et al. 2022. iScience. 25:105004. PubMed
  6. Ramakrishnan RK, et al. 2020. PLoS One. 15:e0242695. PubMed
  7. Xu Q, et al. 2020. J Ovarian Res. 0.597916667. PubMed
  8. Fachi JL, et al. 2020. J Exp Med. 217:00:00. PubMed
  9. Descalzi-Montoya D, et al. 2021. Chembiochem. 22:1589. PubMed
  10. Kumar MP, et al. 2018. Cell Rep. 25:1458. PubMed
  11. Li C, et al. 2022. Cell Mol Gastroenterol Hepatol. 13:1805. PubMed
  12. Li N, et al. 2021. Cell Rep Med. 2:100297. PubMed
  13. Zhang L, et al. 2021. Cell Mol Immunol. 18:230. PubMed
  14. Schedel F, et al. 2020. Pigment Cell Melanoma Res. 33:63. PubMed
  15. Okuma A, et al. 2017. Nat Commun.. 10.1038/s41467-017-02281-x. PubMed
  16. Felker S, et al. 2022. JCI Insight. 7:. PubMed
  17. Ma Y, et al. 2017. Neurochem Res. 10.1007/s11064-017-2207-y. PubMed
  18. DU T, et al. 2020. Front Immunol. 1.571527778. PubMed
  19. Li S, et al. 2020. Onco Targets Ther. 13:12383. PubMed
  20. Sun Y, et al. 2022. iScience. 25:104846. PubMed
  21. Khan N, et al. 2020. Cell. 183:752. PubMed
  22. Xu L, et al. 2022. Front Immunol. 13:841141. PubMed
  23. Howie D, et al. 2019. Front Immunol. 10:1860. PubMed
  24. Fan J, et al. 2021. Front Oncol. 10:574860. PubMed

Antigen Details

Biology Area
Apoptosis/Tumor Suppressors/Cell Death, Cell Biology, Cell Proliferation and Viability, Neuroscience
Gene ID
308 View all products for this Gene ID

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.

What type of PE do you use in your conjugates?
We use R-PE in our conjugates.
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.

Go To Top Version: 4    Revision Date: 10/22/2024

For Research Use Only. Not for diagnostic or therapeutic use.

 

This product is supplied subject to the terms and conditions, including the limited license, located at www.biolegend.com/terms) ("Terms") and may be used only as provided in the Terms. Without limiting the foregoing, BioLegend products may not be used for any Commercial Purpose as defined in the Terms, resold in any form, used in manufacturing, or reverse engineered, sequenced, or otherwise studied or used to learn its design or composition without express written approval of BioLegend. Regardless of the information given in this document, user is solely responsible for determining any license requirements necessary for user’s intended use and assumes all risk and liability arising from use of the product. BioLegend is not responsible for patent infringement or any other risks or liabilities whatsoever resulting from the use of its products.

 

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This data display is provided for general comparisons between formats.
Your actual data may vary due to variations in samples, target cells, instruments and their settings, staining conditions, and other factors.
If you need assistance with selecting the best format contact our expert technical support team.

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