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- Regulatory Status
- RUO
- Other Names
- Annexin A5 Apoptosis Detection Kit
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- Product Citations
- publications
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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- FITC for 15 minutes at 37°C in Annexin V Binding buffer. Propidium Iodide (PI) (cat. 421301 at 0.03 µg/Test) was added 5 minutes prior to running tubes.
BioLegend's FITC 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
- 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
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Store between 2°C and 8°C. Do not freeze.
Caution: Propidium Iodide Solution is toxigenic and mutagenic; handle with care. - Application
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FC - Quality tested
- Recommended Usage
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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 FITC 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. - 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) - Product Citations
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Antigen Details
- Biology Area
- Apoptosis/Tumor Suppressors/Cell Death, Cell Biology, Neuroscience
- Gene ID
- 308 View all products for this Gene ID
Related Pages & Pathways
Pathways
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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