Pacific Blue™ Annexin V Apoptosis Detection Kit with PI

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.

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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

Documentation

• Technical data sheet
• Certificate of Analysis
• Safety Data Sheet

Reviews

Related Pages & Pathways

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.

Certificate of Analysis