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Annexin V-iFluor® 568 conjugate

Product key features

Annexin V-iFluor® 568 Conjugate is a fluorescent probe for detecting early apoptotic cells via phosphatidylserine exposure.

  • Bright & Photostable Fluorophore: Labeled with iFluor® 568 for strong orange-red fluorescence.
  • Early Apoptosis Detection: Binds externalized PS on apoptotic cells before membrane damage or morphological change.
  • Versatile Applications: Ideal for flow cytometry, live-cell imaging, and other apoptosis assays.
  • Channel Compatibility: Easily integrates into Texas Red® or TRITC filter sets for multicolor workflows.

Product description

Annexin V-iFluor® 568 Conjugate is a fluorescent apoptosis detection reagent that combines Annexin V with iFluor® 568, a bright orange-red fluorophore with excitation/emission maxima of ~566/586 nm. This conjugate enables sensitive and direct detection of phosphatidylserine (PS) exposure on the outer plasma membrane—a hallmark of early apoptosis.

By binding PS in a calcium-dependent manner, Annexin V-iFluor® 568 serves as a robust tool for identifying apoptotic cells before morphological changes occur. It is suitable for fluorescence microscopy, flow cytometry, and high-content screening of cell death pathways across a range of research applications.

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 Green™ DCS1 (Cat No. 17550) 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® 633 conjugate64065425000010.2910.0620.044
Annexin V-iFluor® 647 conjugate65667025000010.2510.030.03
Annexin V-iFluor® 680 conjugate68470122000010.2310.0970.094
Annexin V-iFluor® 700 conjugate69071322000010.2310.090.04
Annexin V-iFluor® 750 conjugate75777927500010.1210.0440.039

References

View all 50 references: Citation Explorer
Synthesis, Anticancer Activity, and Mitochondria-targeted Bioimaging Applications of Novel Fluorescent Calix [4]arenes-benzimidazole Derivatives.
Authors: Solmaz, Beyza and Oguz, Alev and Oguz, Mehmet and Ozturk, Bahadir and Yilmaz, Mustafa
Journal: Current medicinal chemistry (2025)
Paclitaxel-induced cognitive decline was attenuated by necroptosis inhibition.
Authors: Liu, Lan-Lan and Zhao, Shuang and Li, Zhao and Li, Hui-Zhou and Ma, Dong-Yang and Liu, Xin and Wang, Gui-Ying and Wang, Xiu-Li
Journal: Neuroreport (2025): 61-69
Cathepsin B Activatable Fluorescent Probe for Antitumor Efficiency Feedback: Attempt To Detect Certain Apoptotic Cells.
Authors: Yu, Qiqi and Xu, Dongdong and Chen, Sina and Yu, Yanlu and Yu, Huanan and Li, Yang and Sun, Wen and Yin, Shouchun
Journal: Analytical chemistry (2025): 2932-2940
Integrated serum pharmacochemistry, network pharmacology and experimental verification to explore the mechanism of Aconiti Lateralis Radix Praeparata in treatment of lung cancer.
Authors: Zhang, Wen and Cai, Shuhui and Luan, Wenhao and Ding, Menglei and Di, Liuqing
Journal: Journal of pharmaceutical and biomedical analysis (2025): 116472
Ethanol extract of Moschus attenuates glutamate-induced cytotoxicity in HT22 cells by regulating the Nrf2 and MAPK pathways.
Authors: Chu, Zhili and Chen, Yubing and Xie, Danni and Song, Caiyou and Yang, Lin and Qin, Tao and Zhai, Zhenwei and Cao, Zhixing and Xu, Ying and Sun, Tao
Journal: Journal of ethnopharmacology (2025): 119879
Page updated on August 10, 2025

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Catalog Number20078
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Physical properties

Solvent

Water

Spectral properties

Correction Factor (260 nm)

0.34

Correction Factor (280 nm)

0.15

Extinction coefficient (cm -1 M -1)

1000001

Excitation (nm)

568

Emission (nm)

587

Quantum yield

0.571

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

Platform

Flow cytometer

Excitation586 nm laser
Emission566 nm
Instrument specification(s)PE or PE-Texas Red

Fluorescence microscope

ExcitationTRITC, Cy3 filter set
EmissionTRITC, Cy3 filter set
Recommended plateBlack wall, clear bottom