logo
AAT Bioquest

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 Pacific Blue® (Pacific Blue® is the trademark of Invitrogen). The blue fluorescent stain is well excited with the Violet Laser at 405 nm, and emits intense blue fluorescence at ~450 nm.

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-mFluor™ Violet 510 conjugate4125052500010.8610.4640.366
Annexin V-mFluor™ Violet 540 conjugate4025351800010.2111.3260.543

Citations

View all 4 citations: Citation Explorer
Tumor-initiating stem cell shapes its microenvironment into an immunosuppressive barrier and pro-tumorigenic niche
Authors: He, Xi and Smith, Sarah E and Chen, Shiyuan and Li, Hua and Wu, Di and Meneses-Giles, Paloma I and Wang, Yongfu and Hembree, Mark and Yi, Kexi and Zhao, Xia and others,
Journal: Cell reports (2021): 109674
White light-induced cell apoptosis by a conjugated polyelectrolyte through singlet oxygen generation
Authors: Liang, Jiamei and Wu, Pan and Tan, Chunyan and Jiang, Yuyang
Journal: RSC advances (2018): 9218--9222
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
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
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
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
Page updated on October 8, 2024

Ordering information

Price
Conjugate
Unit size
Catalog Number
Quantity
Add to cart

Additional ordering information

Telephone1-800-990-8053
Fax1-800-609-2943
Emailsales@aatbio.com
InternationalSee distributors
Bulk requestInquire
Custom sizeInquire
Technical SupportContact us
Purchase orderSend to sales@aatbio.com
ShippingStandard overnight for United States, inquire for international
Request quotation

Physical properties

Molecular weight

~36000

Solvent

Water

Spectral properties

Absorbance (nm)

406

Correction Factor (260 nm)

0.338

Correction Factor (280 nm)

0.078

Extinction coefficient (cm -1 M -1)

350001

Excitation (nm)

406

Emission (nm)

445

Quantum yield

0.811

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

Excitation405 nm laser
Emission450, 40 nm filter
Instrument specification(s)Pacific Blue channel

Fluorescence microscope

ExcitationDAPI filter set
EmissionDAPI filter set
Recommended plateBlack wall, clear bottom
The detection of binding activity of Annexin V-mFluor™ Violet 450 conjugate 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™ Violet 450 conjugate for 30 minutes. Fluorescence intensity was measured using an ACEA NovoCyte flow cytometer in the Pacific Blue channel.
The detection of binding activity of Annexin V-mFluor™ Violet 450 conjugate 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™ Violet 450 conjugate for 30 minutes. Fluorescence intensity was measured using an ACEA NovoCyte flow cytometer in the Pacific Blue channel.
The detection of binding activity of Annexin V-mFluor™ Violet 450 conjugate 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™ Violet 450 conjugate for 30 minutes. Fluorescence intensity was measured using an ACEA NovoCyte flow cytometer in the Pacific Blue channel.