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Pacific Blue™ Annexin V Apoptosis Detection Kit with PI

Pricing & Availability
Regulatory Status
RUO
Other Names
Annexin A5 Apoptosis Detection Kit
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Product Citations
publications
Annexin-V_PB-PI_Antibody_FC_1_050714
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 Pacific Blue™ Annexin V Apoptosis Detection Kit with PI.
  • Annexin-V_PB-PI_Antibody_FC_1_050714
    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 Pacific Blue™ Annexin V Apoptosis Detection Kit with PI.
  • Annexin-V_PB-PI_Antibody_FC_2_050714
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Cat # Size Price Save
640928 100 tests ¥73,700
Description

BioLegend's Pacific Blue™ Annexin V Apoptosis Detection Kit with PI 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 Propidium Iodide Solution (PI). Early apoptotic cells will exclude 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.

Propidium iodide is a fluorescent dye that binds to DNA. When excited by 488 nm laser light, it can be detected with in the PE/Texas Red® channel with a bandpass filter 610/10. It is commonly used in evaluation of cell viability or DNA content in cell cycle analysis by flow cytometry.

Product Details
Technical data sheet

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: Propidium Iodide Solution is toxigenic and mutagenic; handle with care.
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 Pacific Blue™ Annexin V.
4. Add 10 µL of Propidium Iodide 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.


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

View full statement regarding label licenses
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. Ranganathan P, et al. 2014. J AM Soc Nephrol. 25:239. PubMed
Product Citations
  1. Zhang H, et al. 2020. Nat Cancer. 1:826. PubMed
  2. Mehus AA, et al. 2023. Int J Mol Sci. 24:. PubMed
  3. Shyam R, et al. 2022. Front Cell Dev Biol. 10:878395. PubMed
  4. Nudel K, et al. 2015. Infect Immun . 26: 2183-2197. PubMed
  5. Bai Y, et al. 2019. J Biol Chem. 294:9911. PubMed
  6. Gourvest M, et al. 2021. Leukemia. . 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.

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.

 

BioLegend, the BioLegend logo, and all other trademarks are property of BioLegend, Inc. or their respective owners, and all rights are reserved.

 

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Toll-Free Phone: 1-877-Bio-Legend (246-5343) Phone: (858) 768-5800 Fax: (877) 455-9587

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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