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Annexin V, FITC Labeled

Binding activity of FITC-Annexin V conjugate to phosphatidylserine (PS) residues 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 FITC-Annexin V conjugate for 30 minutes. The fluorescence intensity was measured using an ACEA NovoCyte flow cytometer in the FITC channel.
Binding activity of FITC-Annexin V conjugate to phosphatidylserine (PS) residues 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 FITC-Annexin V conjugate for 30 minutes. The fluorescence intensity was measured using an ACEA NovoCyte flow cytometer in the FITC channel.
Binding activity of FITC-Annexin V conjugate to phosphatidylserine (PS) residues 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 FITC-Annexin V conjugate for 30 minutes. The fluorescence intensity was measured using an ACEA NovoCyte flow cytometer in the FITC channel.
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Physical properties
Molecular weight~36000
SolventWater
Spectral properties
Correction Factor (280 nm)0.35
Extinction coefficient (cm -1 M -1)73000
Excitation (nm)491
Emission (nm)516
Quantum yield0.92
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12352200

OverviewpdfSDSpdfProtocol


Molecular weight
~36000
Correction Factor (280 nm)
0.35
Extinction coefficient (cm -1 M -1)
73000
Excitation (nm)
491
Emission (nm)
516
Quantum yield
0.92
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 FITC-Annexin V conjugate is widely used for monitoring cell apoptosis with a flow cytometer or a fluorescence microscope. For microplate assays of annexin V-based cell apoptosis detection, we recommend you use our Cell Meter™ kits.

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

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


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Correction Factor (280 nm)0.35
Extinction coefficient (cm -1 M -1)73000
Excitation (nm)491
Emission (nm)516
Quantum yield0.92

Product Family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Correction Factor (260 nm)Correction Factor (280 nm)
Annexin V, TRITC Labeled5445701000000.270.34

Images


Citations


View all 2 citations: Citation Explorer
The role of non-coding RNA ASBEL regulation of BTG3 in pancreatic ductal adenocarcinoma tumor progression
Authors: Chen, Jing and Zhu, Ming-Yuan and Huang, Yan-Hua and Ling, Yi-Ting and Gu, Tian-Yuan and Zhou, Quan and Fei, Ming-Jian and Zhou, Zhong-Cheng
Journal: Current Therapeutic Research (2023): 100700
Response of Mammalian cells to non-thermal intense narrowband pulsed electric fields
Authors: Katsuki, Sunao and Li, Yulan and Miyakawa, Daiki and Yamada, Ryo and Onishi, Nobuaki and Lim, Soowon
Journal: (2017): 1358--1361

References


View all 90 references: Citation Explorer
Annexin A5-functionalized bimodal lipid-based contrast agents for the detection of apoptosis
Authors: van Tilborg GA, Mulder WJ, Deckers N, Storm G, Reutelingsperger CP, Strijkers GJ, Nicolay K.
Journal: Bioconjug Chem (2006): 741
Annexin V/beta5 integrin interactions regulate apoptosis of growth plate chondrocytes
Authors: Wang W, Kirsch T.
Journal: J Biol Chem (2006): 30848
Myocyte apoptosis during acute myocardial infarction in rats is related to early sarcolemmal translocation of annexin A5 in border zone
Authors: Monceau V, Belikova Y, Kratassiouk G, Robidel E, Russo-Marie F, Charlemagne D.
Journal: Am J Physiol Heart Circ Physiol (2006): H965
Analysis of cycloheximide-induced apoptosis in human leukocytes: Fluorescence microscopy using annexin V/propidium iodide versus acridin orange/ethidium bromide
Authors: Baskic D, Popovic S, Ristic P, Arsenijevic NN.
Journal: Cell Biol Int (2006): 924
In-vivo visualization of radiation-induced apoptosis using (125)I-annexin V
Authors: Watanabe H, Murata Y, Miura M, Hasegawa M, Kawamoto T, Shibuya H.
Journal: Nucl Med Commun (2006): 81
Mapping of treatment-induced apoptosis in normal structures: 99mTc-Hynic-rh-annexin V SPECT and CT image fusion
Authors: Kartachova MS, Valdes Olmos RA, Haas RL, Hoebers FJ, van den Brekel MW, van Z and wijk N, Herk M, Verheij M.
Journal: Eur J Nucl Med Mol Imaging (2006): 893
Non-fusion expression in Escherichia coli: Single-step purification of recombinant human annexin A5 for detection of apoptosis
Authors: Wang F, He XW, Yan HL, Huang JJ, Zhang Y, Jiang L, Gao YJ, Sun SH.
Journal: Protein Expr Purif (2006): 80
Quantum dots based probes conjugated to annexin V for photostable apoptosis detection and imaging
Authors: Le Gac S, Vermes I, van den Berg A.
Journal: Nano Lett (2006): 1863
Lymphoma cells protected from apoptosis by dysregulated bcl-2 continue to bind annexin V in response to B-cell receptor engagement: a cautionary tale
Authors: Holder MJ, Barnes NM, Gregory CD, Gordon J.
Journal: Leuk Res (2006): 77
The impact of altered annexin I protein levels on apoptosis and signal transduction pathways in prostate cancer cells
Authors: Hsiang CH, Tunoda T, Whang YE, Tyson DR, Ornstein DK.
Journal: Prostate (2006): 1413