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
Prepare cells with test compounds (200 µL/sample).
Add Annexin V conjugate assay solution.
Incubate at room temperature for 30-60 minutes.
Analyze with a flow cytometer or a fluorescence microscope.
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 an Annexin V-binding assay buffer: 10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2, pH 7.4.
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.
Centrifuge the cells to get 1-5×105 cells/tube.
Resuspend cells in 200 μL of the Annexin V-binding assay buffer from Step 1.
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.
Incubate at room temperature for 30 to 60 minutes, protected from light.
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.Monitor the fluorescence intensity by using a flow cytometer or a fluorescence microscope.
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.
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).
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.
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.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
Pascal Clerc, Pauline Jeanjean, Nicolas Halalli, Michel Gougeon, Bernard Pipy, Julian Carrey, Daniel Fourmy, Véronique Gigoux. Journal of Controlled Release (2017).
Hanshaw RG, Lakshmi C, Lambert TN, Johnson JR, Smith BD. Chembiochem, 6, 2214. (2005).
Spectrum
Product family
Name | Excitation (nm) | Emission (nm) | Extinction coefficient (cm -1 M -1) | Quantum yield | Correction Factor (260 nm) | Correction Factor (280 nm) |
Annexin V-iFluor® 350 conjugate | 345 | 450 | 200001 | 0.951 | 0.83 | 0.23 |
Annexin V-iFluor® 488 conjugate | 491 | 516 | 750001 | 0.91 | 0.21 | 0.11 |
Annexin V-iFluor® 555 conjugate | 557 | 570 | 1000001 | 0.641 | 0.23 | 0.14 |
Annexin V-iFluor® 594 conjugate | 587 | 603 | 2000001 | 0.531 | 0.05 | 0.04 |
Annexin V-iFluor® 633 conjugate | 640 | 654 | 2500001 | 0.291 | 0.062 | 0.044 |
Annexin V-iFluor® 647 conjugate | 656 | 670 | 2500001 | 0.251 | 0.03 | 0.03 |
Annexin V-iFluor® 680 conjugate | 684 | 701 | 2200001 | 0.231 | 0.097 | 0.094 |
Annexin V-iFluor® 700 conjugate | 690 | 713 | 2200001 | 0.231 | 0.09 | 0.04 |
Annexin V-iFluor® 750 conjugate | 757 | 779 | 2750001 | 0.121 | 0.044 | 0.039 |
References
Authors: Solmaz, Beyza and Oguz, Alev and Oguz, Mehmet and Ozturk, Bahadir and Yilmaz, Mustafa
Journal: Current medicinal chemistry (2025)
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
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
Authors: Zhang, Wen and Cai, Shuhui and Luan, Wenhao and Ding, Menglei and Di, Liuqing
Journal: Journal of pharmaceutical and biomedical analysis (2025): 116472
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