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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. The green fluorescent stain is well excited with the Blue Laser at 488 nm, and emits intense orange fluorescence at ~570 nm. Its emission is much stronger than those of Annexin V-Cy3 with 488 nm excitation.

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

Citations

View all 2 citations: Citation Explorer
Targeted Magnetic Intra-Lysosomal Hyperthermia produces lysosomal reactive oxygen species and causes Caspase-1 dependent cell death
Authors: Clerc, Pascal and Jeanjean, Pauline and Halalli, Nicolas and Gougeon, Michel and Pipy, Bernard and Carrey, Julian and Fourmy, Daniel and Gigoux, Véronique
Journal: Journal of Controlled Release (2017)

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

Absorbance (nm)

553

Correction Factor (260 nm)

0.228

Correction Factor (280 nm)

0.179

Extinction coefficient (cm -1 M -1)

1200001

Excitation (nm)

505

Emission (nm)

564

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 or 532 nm laser
Emission575, 26 nm filter
Instrument specification(s)PE channel

Fluorescence microscope

ExcitationCy3, TRITC filter set
EmissionCy3, TRITC filter set
Recommended plateBlack wall, clear bottom
The detection of binding activity of Annexin V-mFluor™ Blue 570 to phosphatidylserine in Jurkat cells. Jurkat cells were treated without (Green) or with 1 μM staurosporine (Red) at 37 °C for 4 hours and then labeled with Annexin V-mFluor™ Blue 570 conjugate for 30 minutes.
The detection of binding activity of Annexin V-mFluor™ Blue 570 to phosphatidylserine in Jurkat cells. Jurkat cells were treated without (Green) or with 1 μM staurosporine (Red) at 37 °C for 4 hours and then labeled with Annexin V-mFluor™ Blue 570 conjugate for 30 minutes.
The detection of binding activity of Annexin V-mFluor™ Blue 570 to phosphatidylserine in Jurkat cells. Jurkat cells were treated without (Green) or with 1 μM staurosporine (Red) at 37 °C for 4 hours and then labeled with Annexin V-mFluor™ Blue 570 conjugate for 30 minutes.