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Annexin V

Annexins are a family of proteins that bind to phospholipid membranes in the presence of calcium. Annexin V is a valuable tool for studying cell apoptosis. It is used as a probe to detect cells which have expressed phosphatidylserine on the cell surface, a feature found in apoptosis as well as other forms of cell death. There are a variety of parameters that can be used for monitoring cell viability. Annexin V-dye conjugates are widely used to monitor cell apoptosis through measuring the translocation of phosphatidylserine (PS). In apoptosis, PS is transferred to the outer leaflet of the plasma membrane. The appearance of phosphatidylserine on the cell surface is a universal indicator of the initial/intermediate stages of cell apoptosis and can be detected before morphological changes can be observed. This fluorescent Annexin V conjugate has spectral properties similar to Alexa Fluor® 488 (Alexa Fluor® 488 is the trademark of Invitrogen) and FITC.

Example protocol

AT A GLANCE

Protocol Summary
  1. Prepare cells with test compounds (200 µL/sample).

  2. Add Annexin V conjugate assay solution.

  3. Incubate at room temperature for 30-60 minutes.

  4. Analyze with a flow cytometer or a fluorescence microscope.

Storage and Handling Conditions

The fluorescent annexin V conjugates are stored in a PBS buffer solution containing 0.1% bovine serum albumin (BSA) with a pH of 7.4. To ensure their stability, it is recommended that the solutions be stored at a temperature of -20°C and protected from light. Avoid exposing the fluorescent conjugates to repeated freeze-thaw cycles as this can have a negative effect on their integrity. These solutions can be stored for at least 6 months under the recommended conditions.

SAMPLE EXPERIMENTAL PROTOCOL

Prepare and Incubate Cells with Annexin V Conjugate
  1. Prepare an Annexin V-binding assay buffer: 10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2, pH 7.4.

  2. Treat cells with test compounds for a desired period of time (e.g., 4-6 hours for Jurkat cells treated with staurosporine) to induce apoptosis.

  3. Centrifuge the cells to get 1-5×105 cells/tube.

  4. Resuspend cells in 200 μL of the Annexin V-binding assay buffer from Step 1.

  5. Add 2 μL of the Annexin V conjugate to the cells.

    Optional: Add a dead cell stain such as Propidium Iodide (Cat No. 17585) into the cells for necrosis cells.

  6. Incubate at room temperature for 30 to 60 minutes, protected from light.

  7. Add 300 μL of the Annexin V-binding assay buffer (from Step 1) to increase volume before analyzing the cells with
    a flow cytometer or fluorescence microscope.

  8. Monitor the fluorescence intensity by using a flow cytometer or a fluorescence microscope.

Flow Cytometer Protocol
  1. Quantify Annexin V conjugates binding by using a flow cytometer with appropriate filters.

    Note: It is not common to perform Annexin V binding flow cytometric analysis on adherent cells due to the possibility of membrane damage during cell detachment or harvesting. However, previous studies by Casiola-Rosen et al. and van Engelend et al. (refer to Refs 1 and 2) have demonstrated methods for using Annexin V in flow cytometry on adherent cell types.

Fluorescence Microscope Protocol
  1. Pipette the cell suspension from Step 6, rinse 1-2 times with Annexin V-binding assay buffer (Step 1), and then resuspend the cells with the Annexin V-binding assay buffer (Step 1).

  2. Add the cells on a glass slide that is covered with a glass cover slip.

    Note: For adherent cells, it is recommended to grow the cells directly on a cover slip. 

  3. After incubation with Annexin V conjugate (Step 6), rinse 1-2 times with Annexin V-binding assay buffer (Step 1), and add
    Annexin V-binding assay buffer (Step 1) back to the cover slip.

  4. Invert the cover slip on a glass slide and visualize the cells. The cells can also be fixed in 2% formaldehyde after incubation with Annexin V conjugate and visualized under a microscope with the appropriate filter set.

APPENDIX

References
  1. Pascal Clerc, Pauline Jeanjean, Nicolas Halalli, Michel Gougeon, Bernard Pipy, Julian Carrey, Daniel Fourmy, Véronique Gigoux. Journal of Controlled Release (2017).

  2. Hanshaw RG, Lakshmi C, Lambert TN, Johnson JR, Smith BD. Chembiochem, 6, 2214. (2005).

Spectrum

Product family

NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
Annexin V-iFluor® 633 conjugate64065425000010.2910.0620.044
Annexin V-iFluor® 350 conjugate3454502000010.9510.830.23
Annexin V-iFluor® 555 conjugate55757010000010.6410.230.14
Annexin V-iFluor® 594 conjugate58760320000010.5310.050.04
Annexin V-iFluor® 647 conjugate65667025000010.2510.030.03
Annexin V-iFluor® 680 conjugate68470122000010.2310.0970.094
Annexin V-iFluor® 750 conjugate75777927500010.1210.0440.039
Annexin V-iFluor® 700 conjugate69071322000010.2310.090.04

Citations

View all 9 citations: Citation Explorer
Synthesis and Biological Activity of 2-Chloro-8-methoxy-5-methyl-5 H-indolo [2, 3-b] Quinoline for the Treatment of Colorectal Cancer by Modulating PI3K/AKT/mTOR Pathways
Authors: Ma, Yunhao and Zhu, Hongmei and Jiang, Xinrong and Zhou, Zhongkun and Zhou, Yong and Tian, Yanan and Tu, Lixue and Lu, Juan and Niu, Yuqing and Du, Liqian and others,
Journal: ACS Omega (2024)
Deoxynivalenol induces m6A-mediated upregulation of p21 and growth arrest of mouse hippocampal neuron cells in vitro
Authors: Xu, Peirong and Zhao, Yulan and Feng, Yue and Zhao, Mindie and Zhao, Ruqian
Journal: Cell Biology and Toxicology (2024): 1--15
A novel selective estrogen receptor degrader induces cell cycle arrest in breast cancer via ER$\alpha$ degradation and the autophagy-lysosome pathway
Authors: Zhou, Jiawei and Shen, Rong and Liu, Jing and Deng, Xiangping and Xin, Lilan and Zhou, Hai-Bing and Huang, Jian
Journal: Bioorganic \& Medicinal Chemistry (2023): 117235
Quantification of Xanthone and Anthocyanin in Mangosteen Peel by UPLC-MS/MS and Preparation of Nanoemulsions for Studying Their Inhibition Effects on Liver Cancer Cells
Authors: Li, Rui and Inbaraj, Baskaran Stephen and Chen, Bing-Huei
Journal: International Journal of Molecular Sciences (2023): 3934
Biological impact of magnetic nanoparticles
Authors: Lima, Wilmara Vieira
Journal: (2022)

References

View all 32 references: Citation Explorer
Gold fluorescent annexin A5 as a novel apoptosis detection tool
Authors: Kurschus FC, Pal PP, Baumler P, Jenne DE, Wiltschi B, Budisa N.
Journal: Cytometry A (2009): 626
Glycogen synthase kinase-3 and Omi/HtrA2 induce annexin A2 cleavage followed by cell cycle inhibition and apoptosis
Authors: Wang CY, Lin YS, Su WC, Chen CL, Lin CF.
Journal: Mol Biol Cell (2009): 4153
Evaluation of annexin V and Calcein-AM as markers of mononuclear cell apoptosis during human immunodeficiency virus infection
Authors: Palma PF, Baggio GL, Spada C, Silva RD, Ferreira SI, Treitinger A.
Journal: Braz J Infect Dis (2008): 108
Detection of apoptosis induced by new type gosling viral enteritis virus in vitro through fluorescein annexin V-FITC/PI double labeling
Authors: Chen S, Cheng AC, Wang MS, Peng X.
Journal: World J Gastroenterol (2008): 2174
Measurement of annexin V uptake and lactadherin labeling for the quantification of apoptosis in adherent Tca8113 and ACC-2 cells
Authors: Hu T, Shi J, Jiao X, Zhou J, Yin X.
Journal: Braz J Med Biol Res (2008): 750
Page updated on October 8, 2024

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Physical properties

Molecular weight

~36000

Solvent

Water

Spectral properties

Correction Factor (260 nm)

0.21

Correction Factor (280 nm)

0.11

Extinction coefficient (cm -1 M -1)

750001

Excitation (nm)

491

Emission (nm)

516

Quantum yield

0.91

Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22

Storage

Freeze (< -15 °C); Minimize light exposure
UNSPSC12352200

Platform

Flow cytometer

Excitation488 nm laser
Emission530, 30 nm filter
Instrument specification(s)FITC channel

Fluorescence microscope

ExcitationFITC filter set
EmissionFITC filter set
Recommended plateBlack wall, clear bottom
The detection of binding activity of Annexin V-iFluor® 488 and phosphatidylserine in Jurkat cells. Jurkat cells were treated without (Blue) or with 20 &micro;M camptothecin (Red) in a 37 &deg;C, 5% CO2 incubator for 4-5 hours, and then dye loaded with Annexin V-iFluor® 488 for 30 minutes. The fluorescence intensity of Annexin V-iFluor® 488 was measured with a FACSCalibur (Becton Dickinson) flow cytometer using the FL1 channel.
The detection of binding activity of Annexin V-iFluor® 488 and phosphatidylserine in Jurkat cells. Jurkat cells were treated without (Blue) or with 20 &micro;M camptothecin (Red) in a 37 &deg;C, 5% CO2 incubator for 4-5 hours, and then dye loaded with Annexin V-iFluor® 488 for 30 minutes. The fluorescence intensity of Annexin V-iFluor® 488 was measured with a FACSCalibur (Becton Dickinson) flow cytometer using the FL1 channel.
The detection of binding activity of Annexin V-iFluor® 488 and phosphatidylserine in Jurkat cells. Jurkat cells were treated without (Blue) or with 20 &micro;M camptothecin (Red) in a 37 &deg;C, 5% CO2 incubator for 4-5 hours, and then dye loaded with Annexin V-iFluor® 488 for 30 minutes. The fluorescence intensity of Annexin V-iFluor® 488 was measured with a FACSCalibur (Becton Dickinson) flow cytometer using the FL1 channel.
Photostability of Annexin V-iFluor® 488 (Red) and Annexin V-FITC (Blue). Jurkat cells were treated with 1 &micro;M&nbsp; staurosporine in a 37 &deg;C, 5% CO2 incubator for 4-5 hours and then&nbsp;stained with Annexin V conjugate for 30min. The cells were continuously exposed to microscope (Keyence BZ-X710) excitation light source with a&nbsp;FITC filter for 5 min. Images were captured every second for&nbsp;5 minutes, and total fluorescence intensity was normalized to the intenisty at 0 s.&nbsp;