- Regulatory Status
- Other Names
- Annexin A5 Apoptosis Detection Kit
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- Product Citations
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BioLegend's FITC 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).
- All mammalian species
- Lot-specific (please contact technical support for concentration and total µg amount, or use our Lookup tool if you have a lot number.)
- 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.
FC - Quality tested
- Recommended Usage
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 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
0.5 ml of FITC Annexin V
0.5 ml of 7-AAD Viability Staining Solution
50 ml of Annexin V Binding Buffer
Materials Not Included:
Cell Staining Buffer (Cat. No. 420201)
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
- Maciel E, et al. 2014. Arch Biochem Biophys. 548:38. PubMed
- Product Citations
- 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.
- Why is washing not recommended after the addition 7-AAD or PI addition when assessing viability?
These dyes bind in equilibrium with DNA. Therefore, external dye concentration must be maintained during analysis and the dye should not be washed out.
- 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.
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