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Pacific Blue™ Annexin V

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Regulatory Status
RUO
Other Names
Annexin A5
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Product Citations
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PB_Annexin-V_kit_052318
Human T leukemia cell line Jurkat, treated (left) or non-treated (right) with BioLegend’s anti-human CD95 (EOS9.1) mAb (Cat. No. 305704) for 4 hours at 37°C, then stained with Annexin V- Pacific Blue™ for 15 minutes at 37°C in Annexin V Binding buffer. Helix NP Green (Cat. No. 425303 at 1.25 nM) was added 5 minutes at room temperature prior to running tubes.
  • PB_Annexin-V_kit_052318
    Human T leukemia cell line Jurkat, treated (left) or non-treated (right) with BioLegend’s anti-human CD95 (EOS9.1) mAb (Cat. No. 305704) for 4 hours at 37°C, then stained with Annexin V- Pacific Blue™ for 15 minutes at 37°C in Annexin V Binding buffer. Helix NP Green (Cat. No. 425303 at 1.25 nM) was added 5 minutes at room temperature prior to running tubes.
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640917 25 tests 100€
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640918 100 tests 235€
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Description

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 cells and necrotic. 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
Technical Data Sheet (pdf)

Product Details

Verified Reactivity
Human, Mouse, Rat
Reported Reactivity
Other Species
Formulation
Phosphate-buffered solution, pH 7.2, containing 0.09% sodium azide and BSA (origin USA)
Preparation
The purified protein was conjugated with Pacific Blue™ under optimal conditions.
Concentration
Lot-specific (to obtain lot-specific concentration and expiration, please enter the lot number in our Certificate of Analysis online tool.)
Storage & Handling
The Annexin V solution should be stored undiluted between 2°C and 8°C, and protected from prolonged exposure to light. Do not freeze.
Application

FC - Quality tested
Recommended Usage

Each lot of this product is quality control tested by immunofluorescent staining with flow cytometric analysis. For flow cytometric staining, the suggested use of this reagent is 5 µl 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.

* Pacific Blue™ has a maximum emission of 455 nm when it is excited at 405 nm. Prior to using Pacific Blue™ conjugate for flow cytometric analysis, please verify your flow cytometer's capability of exciting and detecting the fluorochrome.


Alexa Fluor® and Pacific Blue™ are trademarks of Life Technologies Corporation.

View full statement regarding label licenses
Excitation Laser
Violet Laser (405 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 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
  1. Gordy C, et al. 2011 Blood 117:618. PubMed
  2. Jia W, et al. 2011. J. Immunol. 186:5313. PubMed
  3. Naegele M, et al. 2012. J. Neuroimmunol. 242:60. PubMed
  4. Jenke AC, et al. 2013. PLoS One. 8:e55636. PubMed
  5. Oh J, et al. 2013. J Exp Med. 210:1069. PubMed
  6. Hasan S, et al. 2013. Blood. 122:1464. PubMed
  7. Zhao BB, et al. 2013. PLoS One. 8:77008. PubMed
  8. Mihaly SR, et al. 2014. PLoS One. 9:94982. PubMed
  9. Miyazawa M, et al. 2014. Mol Biol Cell. 25:2116. PubMed
  10. Yabas M, et al. 2014. J Biol Chem. 289:19531. PubMed
  11. Burbulla LF, et al. 2014. Cell Death Dis. 5:1180. PubMed
Product Citations
  1. Tsuji M 2014. Mol Biol Cell. 25:2116. PubMed
  2. Ocklenburg T, et al. 2021. Sci Rep. 7199:11. PubMed
  3. Uriostegui–Arcos M, et al. 2020. Open Biol. 10:200050. PubMed
  4. Blake D, et al. 2022. Elife. 11:. PubMed
  5. Bujarrabal-Dueso A, et al. 2023. Nat Struct Mol Biol. 30:475. PubMed
  6. He W 2011. J Immunol. 186:5313. PubMed
  7. Lindqvist B, et al. 2022. PLoS Pathog. 18:e1010555. PubMed
  8. Angiari S, et al. 2020. Cell Metab. 31:391. PubMed
  9. Liang F, et al. 2021. Front Immunol. 12:658715. PubMed
  10. Mascarenhas M, et al. 2016. Blood. 127: 2298 - 2309. PubMed
  11. Yin W, et al. 2022. Biomolecules. 12:. PubMed
  12. Jenke A, et al. 2013. PLoS One. 8:e55636. PubMed
  13. Petrillo C, et al. 2018. Cell Stem Cell. 1.527777778. PubMed
  14. Cha SE, et al. 2021. Oncoimmunology. 10:1899469. PubMed
  15. Khan E, et al. 2016. Sci Rep. 6:38104. PubMed
  16. Mihaly S, et al. 2014. PLoS One. 9:94982. PubMed
  17. Martel C, et al. 2011. PLoS One. 6:e18812. PubMed
  18. Saint Fleur–Lominy S, et al. 2018. Cell Rep. 24:3045. PubMed
  19. Gurrion C, et al. 2017. J Cancer. 2.323611111. PubMed
  20. Oh J, et al. 2013. J Exp Med. 210:1069. PubMed
  21. Burbulla L, et al. 2014. Cell Death Dis. 5:1180. PubMed
  22. Peiris T, et al. 2016. Development. 143: 1697 - 1709.. PubMed
  23. Ramstead AG, et al. 2020. Cell Rep. 30:2889. PubMed
  24. Hewitt KJ et al. 2017. Developmental cell. 42(3):213-225 . PubMed
  25. Yabas M, et al. 2016. PLoS One. 11: 0146774. PubMed
  26. Lao Z, et al. 2015. PLoS One. 10: 0133895. PubMed
  27. Khuat LT, et al. 2020. Sci Transl Med. 12:. PubMed
  28. Naegele M, et al. 2012. J Neuroimmunol. 242:60. PubMed
  29. Hejna M, et al. 2017. Sci Rep. 10.1038/s41598-017-12165-1. PubMed
  30. Gordy C, et al. 2011. Blood. 117:618. PubMed
  31. Kiss M, et al. 2020. Cancer Immunol Res. 9:309. PubMed
  32. Hasan S, et al. 2013. Blood. 122:1464. PubMed
  33. Wu Z, et al. 2019. Nat Metab. 1:1209. PubMed
  34. Chen X, et al. 2017. Autophagy. 1.204861111. PubMed
  35. Silginer M, et al. 2017. Cell Death Dis. 10.1038/cddis.2017.171. PubMed
  36. Zhao B, et al. 2013. PLoS One. 8:77708. PubMed
  37. Guentsch A, et al. 2017. Mol Cell Biol. 37: e00236-16. PubMed
  38. Medina CB, et al. 2021. Immunity. 54(8):1715-1727.e7. PubMed
  39. Kranz P, et al. 2017. Cell Death Dis. 8:e2986. PubMed
  40. Bjørnstad R, et al. 2019. Mol Cancer Ther. 1.14375. PubMed
  41. Wang X, et al. 2022. Elife. 11:. PubMed
  42. Terlecki–Zaniewicz L, et al. 2018. Aging (Albany NY). 1.182638889. PubMed
  43. Sun Z, et al. 2022. Cancer Res. . PubMed
  44. Anderson CJ, et al. 2021. Nature. 596:262. PubMed
  45. León TE, et al. 2020. Cancer Discov. 10:998. PubMed
  46. Yabas M, et al. 2014. J Biol Chem. 289:19531. PubMed
  47. Mei Y, et al. 2020. Nat Commun. 2.661111111. PubMed
  48. Fontanals–Cirera B, et al. 2017. Mol Cell. 68:731. PubMed
RRID
AB_1279046 (BioLegend Cat. No. 640917)
AB_1279044 (BioLegend Cat. No. 640918)

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.

Go To Top Version: 7    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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