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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. iFluor 633-Annexin V conjugate has spectral properties similar to Alexa Fluor® 633 with fluorescence excitation and emission maxima of ~638 nm and ~655 nm respectively (Alexa Fluor® is the trademark of Invitrogen).

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 Nuclear Violet™ DCS1 (Cat No. 17549) 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® 350 conjugate3454502000010.9510.830.23
Annexin V-iFluor® 488 conjugate4915167500010.910.210.11
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 4 citations: Citation Explorer
A drug-like imidazole-benzothiazole conjugate inhibits malignant melanoma by stabilizing the c-MYC G-quadruplex
Authors: Wu, Tian-Ying and Huang, Qiong and Huang, Zhi-Shu and Hu, Ming-Hao and Tan, Jia-Heng
Journal: Bioorganic Chemistry (2020): 103866
New substituted quinoxalines inhibit triple-negative breast cancer by specifically downregulating the c-MYC transcription
Authors: Hu, Ming-Hao and Wu, Tian-Ying and Huang, Qiong and Jin, Guangyi
Journal: Nucleic acids research (2019)
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

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Physical properties

Molecular weight

~36000

Solvent

Water

Spectral properties

Correction Factor (260 nm)

0.062

Correction Factor (280 nm)

0.044

Extinction coefficient (cm -1 M -1)

2500001

Excitation (nm)

640

Emission (nm)

654

Quantum yield

0.291

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

Excitation633 or 640 nm laser
Emission660, 20 nm filter
Instrument specification(s)APC channel

Fluorescence microscope

ExcitationCy5 filter set
EmissionCy5 filter set
Recommended plateBlack wall, clear bottom
The detection of binding activity of Annexin V-iFluor® 633 conjugate to phosphatidylserine in Jurkat cells. Jurkat cells were treated without (Green) or with 1 μM staurosporine (Red) in 37 ºC for 4 hours, and then labeled with Annexin V-iFluor® 633 conjugate for 30 minutes. Fluorescence intensity was measured using ACEA NovoCyte flow cytometer in APC channel.
The detection of binding activity of Annexin V-iFluor® 633 conjugate to phosphatidylserine in Jurkat cells. Jurkat cells were treated without (Green) or with 1 μM staurosporine (Red) in 37 ºC for 4 hours, and then labeled with Annexin V-iFluor® 633 conjugate for 30 minutes. Fluorescence intensity was measured using ACEA NovoCyte flow cytometer in APC channel.
The detection of binding activity of Annexin V-iFluor® 633 conjugate to phosphatidylserine in Jurkat cells. Jurkat cells were treated without (Green) or with 1 μM staurosporine (Red) in 37 ºC for 4 hours, and then labeled with Annexin V-iFluor® 633 conjugate for 30 minutes. Fluorescence intensity was measured using ACEA NovoCyte flow cytometer in APC channel.